Oral care compositions and methods of use

ABSTRACT

Disclosed herein are oral care compositions comprising naringin:Zinc complexes having a 2:1 naringin to zinc molar ratio. Methods of making and using the compositions are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application Ser. No.62/412,032, filed on Oct. 24, 2016, the content of which is expresslyincorporated herein by reference.

FIELD

This invention relates to oral care compositions comprisingNaringin:Zinc complexes as well as to methods of using and of makingthese compositions.

BACKGROUND

Naringin is a flavanone glycoside, commonly found in grapefruits andcitrus fruits. It has been documented to exhibit health benefits ondiverse applications, such as antimicrobial activities (Pereira R. etal., Molecules, 2007, 12: 1352-66; Abdulmandi A-H. et al., Acta Chimica& Pharmaceutica Indica, 2015, 5(3): 129-42), wound healing (Kandhare A.D. et al., Chemico-Biological Interactions, 2014, 219: 101-12, PereiraR. et al., supra, Abdulmandi A-H. et al., supra), periodontal diseases,diabetes mellitus and rheumatological disorders (Tsui V. W. K. et al.,Phytotherapy Research, 2008, 22(3): 401-406; Bharti S. et al., PlantaMed, 2014, 80: 437-51). Furthermore, it was demonstrated to inhibit thegrowth of periodontal pathogens as well as some common oralmicroorganisms in vitro (Tsui V. W. K. et al., supra).

Tooth demineralization is a chemical process by which minerals, mainlycalcium, are removed from any of the hard tissues, i.e. enamel, dentine,and cementum (X Li et al., J. of Dentistry, 2014, 42:S12-20). Effects ofdemineralization may be reversed if there is sufficient time to allowremineralization to occur to counteract the acids in the oral cavity.Remineralization is beneficial for the aging population who experiencegum recession as well as patients with severe periodontitis with obviousroot exposure. Remineralization may further offer protection againstcavity progression. A remineralization effect of flavonoids, includingNaringin, on artificial root caries is reported; however; the flavonoidsshowed to be less effective than fluoride (Epasinghe D. J. et al.,Australian Dental Journal, 2016, 61(2):196-202).

Matrix metalloproteinases (MMPs) have been suggested to play animportant role in the destruction of dentine organic matrix followingdemineralization by bacterial acids. Increasing Zinc concentration hasbeen shown to inhibit dentine-MMP dependent collagen degradation(Toledano M. et al., Caries Res., 2012, 46(3):201-207).

Naringin and Zinc citrate have previously been combined in a toothpasteformulation; see CN102218021, published Jan. 23, 2013. Naringin-metalcomplexes for use in insecticide compositions have been described inWO2015027308, published Mar. 3, 2015. Naringin-metal complexes have beenpreviously disclosed (Al-Hassani R. A. et al., Acta Chimica &Pharmaceutica Indica, 2015, 5(3): 129-42); however, synthesis of suchcomplexes utilized ethanol solutions, which is not optimal as aningredient for use in consumer products. Therefore, methods of synthesisare needed for enhanced production of such molecules.

Current oral care market products do not address gum recession benefits.Accordingly, there is a need for oral compositions to treat and/orprevent progression of gum recession.

SUMMARY OF THE INVENTION

It has been surprisingly found that complexes of naringin and zinchaving two moles of naringin to one mole of zinc may be produced. Thesecomplexes show unexpected physical-chemical attributes, such as anincrease in antibacterial activity. Such activity may be useful for usein oral care compositions.

In one embodiment, the invention is a naringin:Zn complex, wherein thenaringin:Zn complex has a 2:1 naringin to zinc molar ratio. In a furtherembodiment, the naringin:Zinc complex has a melting point above 230° C.In further embodiments, the naringin:Zinc complex has a diffusioncoefficient between 2.8e-11 to 3.2e-11 m²/s in DMSO solution at 25° C.

In a particular embodiment, the invention provides an oral carecomposition comprising the naringin:Zinc complex. In certainembodiments, the oral care composition may be selected from the groupconsisting of: a toothpaste or a dentifrice, a mouthwash or a mouthrinse, a topical oral gel and a denture cleanser. In certainembodiments, the oral care composition may be selected from dentalstrips, beads, varnish and toothpowder.

In certain embodiments, a composition of the invention may furthercomprise one or more agents selected from diluents, bicarbonate salts,pH modifying agents, surfactants, foam modulators, additional thickeningagents, humectants, sweeteners, flavorants, pigments, antibacterialagents, anticaries agents, fluoride ion sources, anticalculus or tartarcontrol agents, and mixtures thereof.

In one embodiment, the invention provides a method to improve oralhealth comprising applying an effective amount of the oral compositiondescribed herein to the oral cavity of a subject in need thereof. Incertain embodiments, the oral health may be selected from one or more ofthe following: reduce or inhibit formation of dental caries; reduce,repair or inhibit early enamel lesions; reduce or inhibitdemineralization and promote remineralization of the teeth; reducehypersensitivity of the teeth; reduce or inhibit gingivitis; promotehealing of sores or cuts in the mouth; reduce levels of acid producingbacteria; increase relative levels of arginolytic bacteria; inhibitmicrobial biofilm formation in the oral cavity; raise and/or maintainplaque pH at levels of at least pH 5.5 following sugar challenge; reduceplaque accumulation; treat, relieve or reduce dry mouth; whiten teeth;enhance systemic health, including cardiovascular health; reduce erosionof the teeth; immunize the teeth against cariogenic bacteria and theireffects; clean the teeth and oral cavity; reduce inflammation; andincrease anti-oxidant levels.

In further embodiments, the invention provides for methods for preparinga naringin:Zinc complex having a 2:1 naringin to zinc molar ratio. Incertain embodiments, the method includes a complex preparation stepperformed using a pH between 7-10. In certain embodiments, the methodincludes mixing of naringin and zinc at a temperature between 65-85° C.

In certain embodiments, the method of preparing the 2:1 naringin:zinccomplex comprises the steps of mixing naringin in methanol; adding asource of zinc; adjusting the pH of the solution to 10.0; incubating thereaction; and optionally isolating the complex. In other embodiments,the method comprises the steps of mixing naringin in water; heating themixture to 70° C.; adding a source of zinc; adjusting the pH of thesolution to 10.0; incubating the reaction; and optionally isolating thecomplex. In other embodiments, the method comprises the steps of mixingnaringin in water; heating the mixture to 70° C.; adjusting the pH ofthe solution to 10.0; adding a source of zinc; and optionally isolatingthe complex. In other embodiments, the method comprises the steps ofmixing naringin in water; heating the mixture to 70° C.; adding a sourceof zinc; adjusting the pH of the solution to 7.0; and optionallyisolating the complex. In certain embodiments, the method comprises thesteps of mixing naringin and ZnO in water; heating the mixture;incubating the mixture; and optionally isolating the complex. In certainembodiments, the method comprises mixing naringin in propylene glycol at70° C.; adjusting the pH of the solution to be between 9.0-10.0; addinga source of zinc in propylene glycol at 40-50° C.; and optionallyisolating the complex.

In certain embodiments, the Zn source is selected from zinc acetate,zinc oxide, zinc chloride, zinc lactate, zinc citrate, or zinc nitrate.In certain embodiments, the Zn source is zinc acetate. In certainembodiments, the Zn source is ZnO.

In certain embodiments, the invention is a composition obtained orobtainable by combining the ingredients as set forth in any of thepreceding compositions and methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is the molecular structure of Naringin. FIG. 1B is the proposedmolecular structure for 2:1 naringin:Zinc complex.

FIG. 2 is a UV-VIS spectrum of naringin compared to a 2:1 naringin:Zinccomplex, Product 1, of the invention.

FIG. 3 is a UV-VIS spectrum of naringin compared to various 2:1naringin:Zinc complex products, Products 1-6, of the invention.

FIG. 4 is a UV-VIS spectrum of naringin compared to various 2:1naringin:Zinc complex products, Products 4-6, of the invention.

FIG. 5 is an infrared spectrum of naringin compared with 2:1naringin:Zinc complex, Product 4. The spectra are offset for clarity.Dashed lines are used to bring focus towards differences in that range.

FIG. 6 is a comparison of the infrared vibrational spectra for naringinand complexes of Product 2, Product 4 and Product 5. The spectra areoffset for clarity.

FIG. 7A shows a schematic of the Zinc ion coordinating with the hydroxylgroup on the C5 of naringin. FIG. 7B is a ¹H NMR spectra of naringin and2:1 naringin:Zn complexes (Products 1-6) in DMSO-d5. The chemical shiftvalues reported were referred to TMS as zero. Upper inlet shows proposedstructural nomenclature of the 2:1 naringin:Zn complex.

FIG. 8 is a bar graph showing the experimentally derived diffusioncoefficient of naringin and 2:1 naringin:Zinc complexes (Product 4 andProduct 5) in DMSO-d5.

FIG. 9 is a UV-Vis spectra comparison of naringin and 2:1 naringin:Zinccomplex, Product 5, at week 0 and at week 6.

FIG. 10 is a UV-Vis spectra comparison of naringin and 2:1 naringin:ZincComplex, Product 3, at week 0 and at week 8.

FIG. 11 is a dose response curve comparison of 2:1 naringin:Zinc complex(Product 5), naringin+Zinc acetate combination, Zinc Acetate alone andnaringin alone for Streptococcus sobrinus.

FIG. 12 shows the antimicrobial activity of naringin alone, 2:1naringin:Zinc complex (Product 5), naringin+Zinc acetate combination,and Zinc Acetate alone on single cultures of S. gordonii (S.g), S.sobrinus (S.s) or as co-cultures of S. gordonii and S. sobrinus.

FIG. 13 shows the antimicrobial activity of naringin alone, 2:1naringin:Zinc complex (Product 5), naringin+Zinc acetate combination,and Zinc Acetate alone on single cultures of S.gordonii (S.g), A.viscosus (A.v) or as co-cultures of S. gordonii and A. viscosus.

FIG. 14 shows the antimicrobial activity of naringin alone, 2:1naringin:Zinc complex (Product 5), naringin+Zinc acetate combination,and Zinc Acetate alone on single cultures of S. sobrinus (S.s), A.viscosus (A.v) or as co-cultures of S. sobrinus and A. viscosus.

FIG. 15 shows the antimicrobial activity of naringin alone, 2:1naringin:Zinc complex (Product 5), naringin+Zinc acetate combination,and Zinc Acetate alone on single cultures of A. actinomycetemcomitans(A.a), A. viscosus (A.v) or as co-cultures of A. actinomycetemcomitansand A. viscosus.

FIG. 16 shows the antimicrobial activity of naringin alone, 2:1naringin:Zinc complex (Product 5), naringin+Zinc acetate combination,and Zinc Acetate alone on single cultures of S. sobrinus (S.s) and A.actinomycetemcomitans (A.a) or as co-cultures of S. sobrinus and A.actinomycetemcomitans.

FIG. 17 shows the antimicrobial activity of naringin alone, 2:1naringin:Zinc complex (Product 5), naringin+Zinc acetate combination,and Zinc Acetate alone on mixed bacterial cultures at 37° C., 5% CO₂,anaerobic conditions (OD₆₁₀ 0.1).

FIG. 18 shows the antimicrobial activity of naringin alone, 2:1naringin:Zinc complex (Product 5), naringin+Zinc acetate combination,and Zinc Acetate alone on mixed bacterial cultures at 37° C., aerobicconditions (OD₆₁₀ 0.1).

FIG. 19 is a comparison of zinc update into soft-tissue when treatedwith 2:1 naringin:Zinc complex (Product 5), naringin+Zinc acetatecombination, Zinc Acetate alone or vehicle alone. *−p<0.005.

FIG. 20 is a comparison of the effects of various treatments in cellmigration/proliferation “wound healing” in a scratch assay using HaCatcells.

FIG. 21 is a diagram showing the crossed-shaped image created in thecell monolayer to simulate a wound.

FIG. 22 is a dose response curve comparison of 2:1 naringin:Zinc complex(Product 5), naringin+Zinc acetate combination, Zinc Acetate alone andnaringin alone on a mixed culture grown under anaerobic conditions.

DETAILED DESCRIPTION

The following description of embodiment(s) of the invention is merelyexemplary in nature and is in no way intended to limit the invention,its application, or uses.

As used herein, the words “preferred” and “preferably” refer toembodiments of the invention that afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred, underthe same or other circumstances. Furthermore, the recitation of one ormore preferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

As used throughout, ranges are used as shorthand for describing each andevery value that is within the range. Any value within the range can beselected as the terminus of the range.

Unless stated otherwise, all percentages of composition components givenin this specification are by weight based on a total composition orformulation weight of 100%.

All references cited herein are hereby incorporated by reference intheir entireties. In the event of a conflict in a definition in thepresent disclosure and that of a cited reference, the present disclosurecontrols.

As used herein, the term “oral composition” means the total compositionthat is delivered to the oral surfaces. The composition is furtherdefined as a product which, during the normal course of usage, is notfor the purposes of systemic administration of particular therapeuticagents, intentionally swallowed but is rather retained in the oralcavity for a time sufficient to contact substantially all of the dentalsurfaces and/or oral tissues for the purposes of oral activity. Examplesof such compositions include, but are not limited to, toothpaste or adentifrice, a mouthwash or a mouth rinse, a topical oral gel, a denturecleanser, dental strips, beads, varnish, toothpowder and the like.

As used herein, the term “dentifrice” means paste, gel, or liquidformulations unless otherwise specified. The dentifrice composition canbe in any desired form such as deep striped, surface striped,multi-layered, having the gel surrounding the paste, or any combinationthereof. Alternatively, the oral composition may be dual phase dispensedfrom a separated compartment dispenser.

The term “mouthrinse” in the present invention refers to oralcompositions that are substantially liquid in character, such as a mouthwash, spray, or rinse. In such a preparation the orally acceptablecarrier typically has an aqueous phase comprising water or a water andalcohol mixture. Further, in various embodiments, the oral carrierincludes a humectant and surfactant as described below. Generally, theweight ratio of water to alcohol is in the range of in an amount of 1:1to 20:1, preferably 3:1 to 10:1 and more preferably 4:1 to 6:1. Thetotal amount of water-alcohol mixture in this type of preparation istypically in an amount of 70 to 99.9% of the preparation. In variousembodiments, the alcohol is typically ethanol or isopropanol.

The term “effective amount” as used herein means that the amount of thecomposition of the present invention is of sufficient quantity toachieve the intended purpose, such as, for example, to induce or causeremineralization in the subject.

The terms “2:1 naringin:Zn”, “2:1 naringin:Zn complex”, “naringin:zinc”and “naringin:zinc complex” are used interchangeably and refer, unlessspecified otherwise, to the complex of the invention.

Unless stated otherwise, all percentages of composition components givenin this specification are by weight based on a total composition orformulation weight of 100%.

Unless otherwise specifically identified, the ingredients for use in thecompositions and formulations of the present invention are preferablycosmetically acceptable ingredients. By “cosmetically acceptable” ismeant suitable for use in a formulation for topical application to humanskin. A cosmetically acceptable excipient, for example, is an excipientwhich is suitable for external application in the amounts andconcentrations contemplated in the formulations of this invention, andincludes for example, excipients which are “Generally Recognized asSafe” (GRAS) by the United States Food and Drug Administration.

The compositions and formulations as provided herein are described andclaimed with reference to their ingredients, as is usual in the art. Aswould be evident to one skilled in the art, the ingredients may in someinstances react with one another, so that the true composition of thefinal formulation may not correspond exactly to the ingredients listed.Thus, it should be understood that the invention extends to the productof the combination of the listed ingredients.

The invention provides for compositions comprising naringin:Zncomplex(es), wherein the naringin:Zn complex has a 2:1 naringin to zincmolar ratio. Such complexes provide unique features, such as enhancedantimicrobial activities, useful in oral care applications.

In some embodiments, the present invention provides complexes ofnaringin:zinc having a melting point of at least 200° C., 205° C., 210°C., 215° C., 220° C., 225° C., 230° C., 235° C., 240° C., 245° C., or250° C. In some embodiments, the present invention provides complexes ofnaringin:zinc having a melting point of about 200° C., 205° C., 210° C.,215° C., 220° C., 225° C., 230° C., 235° C., 240° C., 245° C., or 250°C.

The invention further provides for methods of preparing a naringin:Zncomplex having a 2:1 naringin to zinc molar ratio. In certain preferredembodiments, the naringin and zinc source are combined at a pH between7-10. In certain embodiments, the naringin and zinc source are combinedat a pH between 8-10. In certain embodiments, the naringin and zincsource are combined at a pH between 9-10. In certain embodiments, thenaringin and zinc source are combined at a pH of about 7. In certainembodiments, the naringin and zinc source are combined at a pH of about8. In certain embodiments, the naringin and zinc source are combined ata pH of about 9. In certain embodiments, the naringin and zinc sourceare combined at a pH of about 10.

In certain embodiments, the complex is synthesized at a temperaturebetween 20° C. to 80° C. In certain embodiments, the complex issynthesized at a temperature between 25° C. to 75° C. In certainembodiments, the complex is synthesized at a temperature between 30° C.to 70° C. In certain embodiments, the complex is synthesized at atemperature between 35° C. to 65° C. In certain embodiments, the complexis synthesized at a temperature between 20° C. to 40° C. In certainembodiments, the complex is synthesized at a temperature between 40° C.to 60° C. In certain embodiments, the complex is synthesized at atemperature between 60° C. to 80° C. In certain embodiments, the complexis synthesized at a temperature between 20° C. to 25° C.

In certain embodiments, the complex is synthesized at a temperaturebetween 20° C. to 80° C. and a pH between 7-10. In certain embodiments,the complex is synthesized at a temperature between 25° C. to 75° C. anda pH between 7-10. In certain embodiments, the complex is synthesized ata temperature between 30° C. to 70° C. and a pH between 7-10.

In certain embodiments, the method of preparing the 2:1 naringin:zinccomplex comprises the steps of mixing naringin in methanol; adding asource of zinc; adjusting the pH of the solution to about 10.0;incubating the reaction; and optionally isolating the complex. In otherembodiments, the method comprises the steps of mixing naringin in water;heating the mixture to about 70° C.; adding a source of zinc; adjustingthe pH of the solution to about 10.0; incubating the reaction; andoptionally isolating the complex. In other embodiments, the methodcomprises the steps of mixing naringin in water; heating the mixture toabout 70° C.; adjusting the pH of the solution to about 10.0; adding asource of zinc; and optionally isolating the complex. In otherembodiments, the method comprises the steps of mixing naringin in water;heating the mixture to about 70° C.; adding a source of zinc; adjustingthe pH of the solution to about 7.0; and optionally isolating thecomplex. In certain embodiments, the method comprises the steps ofmixing naringin and ZnO in water; heating the mixture; incubating themixture; and optionally isolating the complex. In certain embodiments,the method comprises mixing naringin in propylene glycol at about 70°C.; adjusting the pH of the solution to be between 9.0-10.0; adding asource of zinc in propylene glycol at about 40-50° C.; and optionallyisolating the complex.

The invention further provides the use of a composition comprising the2:1 naringin:zinc complex to reduce and/or inhibit acid erosion of theenamel, reducing or inhibiting gum recession, controlling microbialgrowth, clean the teeth, reduce bacterially-generated biofilm andplaque, reduce gingivitis, inhibit tooth decay and formation ofcavities, and/or reduce dentinal hypersensitivity. The inventionprovides the use of a composition comprising the 2:1 naringin:Zinccomplex for wound healing. The invention further provides for use of acomposition comprising the 2:1 naringin:Zinc complex for amelioratingand/or preventing inflammation. The invention further provides for useof a composition comprising the 2:1 naringin:Zinc complex forameliorating and/or preventing bleeding.

In certain embodiments, naringin:zinc complexes are included in oralcare compositions. In some embodiments, the oral care composition may beselected from the group selected from a toothpaste or a dentifrice, amouthwash or a mouth rinse, a topical oral gel and a denture cleanser.In some embodiments, the oral care composition may be toothpaste or adentifrice. In some embodiments, the oral care composition may be amouthwash or a mouth rinse. In some embodiments, the oral carecomposition may be a topical oral gel and a denture cleanser.

In further embodiments, the invention is a method to improve oral healthcomprising applying an effective amount of an oral composition describedherein to the oral cavity of a subject in need thereof. In certainembodiments, the method includes use of a 2:1 naringin:zinc complexcomposition selected from the group consisting of a toothpaste or adentifrice, a mouthwash or a mouth rinse, a topical oral gel and adenture cleanser.

The invention further provides methods to reduce and inhibit aciderosion of the enamel, reducing or inhibiting gum recession, controllingmicrobial growth, clean the teeth, reduce bacterially-generated biofilmand plaque, reduce gingivitis, inhibit tooth decay and formation ofcavities, and reduce dentinal hypersensitivity, comprising applying aneffective amount of a composition of the invention, e.g., any of saidcompositions described herein, to the teeth.

For example, the invention provides methods to reduce and inhibit aciderosion of the enamel, reducing or inhibiting gum recession, controllingmicrobial growth, clean the teeth, reduce bacterially-generated biofilmand plaque, reduce gingivitis, inhibit tooth decay and formation ofcavities, and reduce dentinal hypersensitivity, comprising applying aneffective amount of a composition of the invention, e.g., any of saidcompositions described herein, to the oral cavity, and then rinsing withsufficient water or aqueous solution.

In certain embodiments, the complex is included in a mouthwash. In someembodiments, the mouthwash comprises from 0.002% to 4% zinc, by weight.In some embodiments, the mouthwash comprises from 0.005% to 0.01% zinc,by weight. In some embodiments, the mouthwash comprises from 0.01% to 1%zinc, by weight. In some embodiments, the mouthwash comprises from 1% to4% zinc, by weight. In some embodiments, the mouthwash of any of theforegoing has zinc solubilized in the formulation, which provides a zincsource upon mixing with a naringin containing solution upon use anddilution with saliva and/or rinsing. In other embodiments, the zinc ionsource and the naringin source form a naringin:zinc complex.

In some embodiments, the pH of the mouthwash is from pH 4 to pH 8.

Some embodiments further comprise an effective amount of a fluoride ionsource within the composition.

In other embodiments, the invention comprises an orally acceptable basecomprising ingredients selected from one or more of buffering agents,humectants, surfactants, thickeners, breath fresheners, flavoring,fragrance, coloring, antibacterial agents, whitening agents, agents thatinterfere with or prevent bacterial attachment, calcium sources,phosphate sources, orally acceptable potassium salts, and anionicpolymers.

Some embodiments provide a mouthwash for use in reducing or inhibitingacid erosion of the enamel, reducing or inhibiting gum recession,controlling microbial growth, cleaning the teeth, reducingbacterially-generated biofilm and plaque, reducing gingivitis,inhibiting tooth decay and formation of cavities, and/or reducingdentinal hypersensitivity.

Some embodiments provide the use of a 2:1 naringin:zinc complex for themanufacture of a mouthwash. Other embodiments provide a method oftreating or reducing dental enamel erosion cleaning the teeth, reducingor inhibiting gum recession, reducing bacterially-generated biofilm andplaque, reducing gingivitis, inhibiting tooth decay and formation ofcavities, and/or reducing dentinal hypersensitivity comprising applyinga mouthwash as described herein. Other embodiments provide methodsfurther comprising the step of rinsing with sufficient water or aqueoussolution.

The invention further provides a method of making an oral carecomposition comprising combining naringin and a zinc source in anaqueous medium, optionally isolating the complex thus formed in solidform, and combining the naringin:zinc complex with an oral carecomposition. In certain embodiments, the oral care composition is atoothpaste. In certain embodiments, the oral care is a mouthwash base.

In various embodiments, the invention provides methods to (i) reducehypersensitivity of the teeth, (ii) reduce plaque accumulation, (iii)reduce or inhibit demineralization and promote remineralization of theteeth, (iv) inhibit microbial biofilm formation in the oral cavity, (v)reduce or inhibit gingivitis, (vi) promote healing of sores or cuts inthe mouth, (vii) reduce levels of acid producing bacteria, (viii)increase relative levels of non-cariogenic and/or non-plaque formingbacteria, (ix) reduce or inhibit formation of dental caries, (x),reduce, repair or inhibit pre-carious lesions of the enamel, e.g., asdetected by quantitative light-induced fluorescence (QLF) or electricalcaries measurement (ECM), (xi) treat, relieve or reduce dry mouth, (xii)clean the teeth and oral cavity, (xiii) reduce erosion, (xiv) whitenteeth; (xv) reduce tartar build-up, and/or (xvi) promote systemichealth, including cardiovascular health, e.g., by reducing potential forsystemic infection via the oral tissues, comprising applying any of saidcompositions described herein to the oral cavity of a person in needthereof, e.g., one or more times per day. In further embodiments, theinvention provides methods to reduce or inhibit gum recession. Infurther embodiments, the invention provides methods to control microbialgrowth. In further embodiments, the invention provides methods toreducing bacterially-generated biofilm, malodor and/or F plaque. Theinvention further provides any of the compositions described herein foruse in any of these methods.

“Actives,” means compounds that, when applied to a target tissue,provide a benefit or improvement to the target tissue. The actives canbe delivered in the form of any oral care formulations, for example atoothpaste, transparent paste, gel, mouthwash, powder, cream, strip,spray, gum, or any other known in the art.

If the complex is delivered in the form of a mouthwash, a persondesiring the benefits rinses with the solution containing thenaringin:zinc complex. In certain embodiments, a dual chamber may beimplemented. In such aspects, a first chamber contains naringin insolution at a basic pH. In certain embodiments, the pH is between 7-10.In certain embodiments, the pH is between 7-8. In certain embodiments,the pH is between 8-9. In certain embodiments, the pH is between 9-10.The dual chamber will also contain a second chamber containing asolubilized zinc source. Upon application, the contents of the first andsecond chamber are mixed together, thus producing the naringin:Zinccomplex.

In another embodiment, the mixture is prepared and immediatelytransferred into a retaining tray, such as those used in holdingwhitening gels, and the person can wear the tray for the effectiveperiod of time. The teeth that are in contact with the mixture will betreated. For use with retaining tray, the mixture can be in the form ofa low-viscosity liquid or a gel. In certain embodiments, the complex isformulated in a composition comprising Carbopol® polymer, glycerin andwater.

In another embodiment, the stock solution, or a mixture of stocksolution with water, is applied to the teeth in a gel formulation, e.g.,wherein the gel can stay on the tooth for an extended period of time foreffective treatment.

In another embodiment, the composition of the present invention is aviscous liquid, preferably a gel, which maintains its consistency duringstorage enabling the product to be painted on the tooth surface with asoft applicator pen or brush. Some embodiments provide a methodutilizing an applicator to deliver the composition, wherein theapplicator is a pen and the pen is stored within an oral care implement.In some embodiments, the pen is removed from the oral care implementprior to application of the composition to the tooth. In someembodiments, the composition is applied to the tooth after brushing. Insome embodiments, the composition is applied to the tooth after brushingwith the oral care implement.

The zinc ion source for complex synthesis may be from any source thatprovides Zn²⁺ ions efficiently, for example zinc oxide, zinc acetate,zinc chloride, zinc lactate, tetrabasic zinc chloride, zinc carbonate,zinc nitrate, zinc citrate, zinc bis lysinate, and zinc phosphate. Zincoxide is a white powder, insoluble in water. Tetrabasic zinc chloride(TBZC) or zinc chloride hydroxide monohydrate is a zinc hydroxy compoundwith the formula Zn₅(OH)₈Cl₂.H₂O, also referred to as basic zincchloride, zinc hydroxychloride, or zinc oxychloride. It is a colorlesscrystalline solid insoluble in water. Both of these materials may besolubilized in water in the presence of naringin and heat, thusproviding a source of zinc ions. In certain preferred embodiments, theZn source is selected from zinc acetate, zinc oxide, zinc chloride, zinclactate, zinc citrate, or zinc nitrate.

In certain embodiments, the amount of zinc in the composition is 0.005to 30% by weight of the composition. In certain embodiments, precursors,e.g., zinc sources and naringin, are present in amounts such that whencombined into the naringin:zinc complex, the complex would be present inan amount of 0.005 to 10% by weight of the composition. In either ofthese embodiments, the amount of the 2:1 naringin:zinc complex can bevaried for the desired purpose, such as a dentifrice or a mouthwash. Inother embodiments, the amount of the 2:1 naringin:zinc complex is atleast 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, atleast 1, at least 2, at least 3, or at least 4 up to 10% by weight ofthe composition. In other embodiments, the amount of the 2:1naringin:zinc complex is less than 9, less than 8, less than 7, lessthan 6, less than 5, less than 4, less than 3, less than 2, less than 1,less than 0.5 to 0.005% by weight of the composition. In otherembodiments, the amounts are 0.05 to 5%, 0.05 to 4%, 0.05 to 3%, 0.05 to2%, 0.1 to 5%, 0.1 to 4%, 0.1 to 3%, 0.1 to 2%, 0.5 to 5%, 0.5 to 4%,0.5 to 3%, or 0.5 to 2% by weight of the composition.

In certain embodiments, the composition is anhydrous. By anhydrous,there is less than 5% by weight water, optionally less than 4, less than3, less than 2, less than 1, less than 0.5, less than 0.1 down to 0% byweight water.

When provided in an anhydrous composition, precursors, e.g., naringinand zinc sources, will not significantly react to form the 2:1naringin:zinc complex. When contacted with a sufficient amount of water,which can be in the form of saliva and/or water used to rinse the mouthduring or after application of the composition, the precursors will thenreact to form the 2:1 naringin:zinc complex. In preferred embodiments,the water and/or saliva has a pH between 7-10. In certain embodiments,the pH is between 7-9. In certain embodiments, the pH is between 7-8.

In certain embodiments, oral care compositions having naringin:zinccomplexes further comprise one or more agents selected from diluents,bicarbonate salts, pH modifying agents, surfactants, foam modulators,additional thickening agents, humectants, sweeteners, flavorants,pigments, antibacterial agents, anticaries agents, fluoride ion sources,anticalculus or tartar control agents, and mixtures thereof.

The oral composition according to the present invention may optionallyinclude other materials, such as for example, cleaning agents,flavouring agents, sweetening agents, adhesion agents, surfactants, foammodulators, abrasives, pH modifying agents, humectants, moisturizers,mouth feel agents, colorants, abrasives, preservatives, fluoride ionsource, saliva stimulating agents, emollients, viscosity modifiers,diluents, emulsifiers, nutrients and combinations thereof. Variouscomponents that may be added to the oral composition include, forexample, a sweetening agent such as saccharin, or sodium saccharin,alcohols such as ethanol, fluoride ion sources such as sodium fluoride,as well as glycerine, sorbitol, polyethylene glycols. Poloxamer polymerssuch as POLOXOMER® 407, PLURONIC® F108, (both available from BASFCorporation), alkyl polyglycoside (APG), polysorbate, PEG40, castor oil,menthol, and the like. It is understood that while general attributes ofeach of the above categories of materials may differ, there may be somecommon attributes and any given material may serve multiple purposeswithin two or more of such categories of materials. Preferably, suchcarrier materials are selected for compatibility with the activeingredients found in magnolia extract or synthetic analogues thereof, aswell as with other ingredients of the composition.

Flavorants among those useful herein include any material or mixture ofmaterials operable to enhance the taste of the composition. Any orallyacceptable natural or synthetic. flavorant can be used, such asflavoring oils, flavoring aldehydes, esters, alcohols, similarmaterials, and combinations thereof. Flavorants include vanillin, sage,marjoram, parsley oil, spearmint oil, cinnamon oil, oil of wintergreen(methylsalicylate) peppermint oil, clove oil, bay oil, anise oil,eucalyptus oil, citrus oils, fruit oils and essences including thosederived from lemon, orange, lime, grapefruit, apricot, banana, grape,apple, strawberry, cherry, pineapple, etc., bean- and nut-derivedflavors such as coffee, cocoa, cola, peanut, almond, etc., adsorbed andencapsulated flavorants, and mixtures thereof. Also encompassed withinflavorants herein are ingredients that provide fragrance and/or othersensory effect in the mouth, including cooling or warming effects. Suchingredients include menthol, menthyl acetate, menthyl lactate, camphor,eucalyptus oil, eucalyptol, anethole, eugenol, cassia, oxanone,[alpha]-irisone, propenyl guaiethol, thymol, linalool, benzaldehyde,cinnamaldehyde, N-ethyl-p-menthan-3-carboxamine,N,2,3-trimethyl-2-isopropylbutanamide, 3-1-menthoxypropane-1,2-diol,cinnamaldehyde glycerol acetal (CGA), methane glycerol acetal (MGA) andmixtures thereof. One or more flavorants are optionally present in atotal amount of 0.01% to 5%, optionally in various embodiments from 0.05to 2%, from 0.1% to 2.5%, and from 0.1 to 0.5%.

Sweetening agents among those useful herein include dextrose,polydextrose, sucrose, maltose, dextrin, dried invert sugar, mannose,xylose, ribose, fructose, levulose galactose, corn syrup, partiallyhydrolyzed starch, hydrogenated starch hydrolysate, sorbitol, mannitol,xylitol, maltitol, isomalt, aspartame, neotame, saccharin and saltsthereof, sucralose, dipeptide-based intense sweeteners, cyclamates,dihydrochalcones, and mixtures thereof.

Mouth-feel agents include materials imparting a desirable texture orother feeling during use of the composition of the invention.

Colorants among those useful herein include pigments, dyes, lakes andagents imparting a particular luster or reflectivity such as pearlingagents. In various embodiments, colorants are operable to provide awhite or light-colored coating on a dental surface, to act as anindicator of locations on a dental surface that have been effectivelycontacted by the composition, and/or to modify appearance, in particularcolor and/or opacity, of the composition to enhance attractiveness tothe consumer. Any orally acceptable colorant can be used, including FD&Cdyes and pigments, talc, mica, magnesium carbonate, calcium carbonate,magnesium silicate, magnesium aluminum silicate, silica, titaniumdioxide, zinc oxide, red, yellow, brown and black iron oxides, ferricammonium ferrocyanide, manganese violet, ultramarine, titaniated mica,bismuth oxychloride, and mixtures thereof. One or more colorants areoptionally present in a total amount of 0.001% to 20%, for example 0.01%to 10% or 0.1% to 5%.

Active Agents:

The compositions of the invention may comprise various agents which areactive to protect and enhance the strength and integrity of the enameland tooth structure and/or to reduce bacteria and associated tooth decayand/or gum disease, including or in addition to the zinc-aminoacid-halide complexes. Effective concentration of the active ingredientsused herein will depend on the particular agent and the delivery systemused. It is understood that a toothpaste for example will typically bediluted with water upon use, while a mouth rinse typically will not be.Thus, an effective concentration of active in a toothpaste willordinarily be 5-15× higher than required for a mouth rinse. Theconcentration will also depend on the exact salt or polymer selected.For example, where the active agent is provided in salt form, thecounterion will affect the weight of the salt, so that if the counterionis heavier, more salt by weight will be required to provide the sameconcentration of active ion in the final product. Arginine, wherepresent, may be present at levels from, e.g., about 0.1 to about 20weight % (expressed as weight of free base), e.g., about 1 to about 10weight % for a consumer toothpaste or about 7 to about 20 weight % for aprofessional or prescription treatment product. Fluoride where presentmay be present at levels of, e.g., about 25 to about 25,000 ppm, forexample about 750 to about 2,000 ppm for a consumer toothpaste, or about2,000 to about 25,000 ppm for a professional or prescription treatmentproduct. Levels of antibacterial agents will vary similarly, with levelsused in toothpaste being e.g., about 5 to about 15 times greater thanused in mouthrinse. For example, a triclosan toothpaste may containabout 0.3 weight % triclosan.

Fluoride Ion Source:

The oral care compositions may further include one or more fluoride ionsources, e.g., soluble fluoride salts. A wide variety of fluorideion-yielding materials can be employed as sources of soluble fluoride inthe present compositions. Examples of suitable fluoride ion-yieldingmaterials are found in U.S. Pat. No. 3,535,421, to Briner et al.; U.S.Pat. No. 4,885,155, to Parran, Jr. et al. and U.S. Pat. No. 3,678,154,to Widder et al. Representative fluoride ion sources include, but arenot limited to, stannous fluoride, sodium fluoride, potassium fluoride,sodium monofluorophosphate, sodium fluorosilicate, ammoniumfluorosilicate, amine fluoride, ammonium fluoride, and combinationsthereof. In certain embodiments the fluoride ion source includesstannous fluoride, sodium fluoride, sodium monofluorophosphate as wellas mixtures thereof. In certain embodiments, the oral care compositionof the invention may also contain a source of fluoride ions orfluorine-providing ingredient in amounts sufficient to supply about 25ppm to about 25,000 ppm of fluoride ions, generally at least about 500ppm, e.g., about 500 to about 2000 ppm, e.g., about 1000 to about 1600ppm, e.g., about 1450 ppm. The appropriate level of fluoride will dependon the particular application. A toothpaste for general consumer usewould typically have about 1000 to about 1500 ppm, with pediatrictoothpaste having somewhat less. A dentifrice or coating forprofessional application could have as much as about 5,000 or even about25,000 ppm fluoride. Fluoride ion sources may be added to thecompositions of the invention at a level of about 0.01 weight % to about10 weight % in one embodiment or about 0.03 weight % to about 5 weight%, and in another embodiment about 0.1 weight % to about 1 weight % byweight of the composition in another embodiment. Weights of fluoridesalts to provide the appropriate level of fluoride ion will obviouslyvary based on the weight of the counterion in the salt.

Foaming Agents:

The oral care compositions of the invention also may include an agent toincrease the amount of foam that is produced when the oral cavity isbrushed. Illustrative examples of agents that increase the amount offoam include, but are not limited to polyoxyethylene and certainpolymers including, but not limited to, alginate polymers. Thepolyoxyethylene may increase the amount of foam and the thickness of thefoam generated by the oral care carrier component of the presentinvention. Polyoxyethylene is also commonly known as polyethylene glycol(“PEG”) or polyethylene oxide. The polyoxyethylenes suitable for thisinvention will have a molecular weight of about 200,000 to about7,000,000. In one embodiment the molecular weight will be about 600,000to about 2,000,000 and in another embodiment about 800,000 to about1,000,000. Polyox® is the trade name for the high molecular weightpolyoxyethylene produced by Union Carbide. The polyoxyethylene may bepresent in an amount of about 1% to about 90%, in one embodiment about5% to about 50% and in another embodiment about 10% to about 20% byweight of the oral care carrier component of the oral care compositionsof the present invention. Where present, the amount of foaming agent inthe oral care composition (i.e., a single dose) is about 0.01 to about0.9% by weight, about 0.05 to about 0.5% by weight, and in anotherembodiment about 0.1 to about 0.2% by weight.

Tartar Control Agents:

In various embodiments of the present invention, the compositionscomprise an anticalculus (tartar control) agent. Suitable anticalculusagents include without limitation phosphates and polyphosphates (forexample pyrophosphates), polyaminopropanesulfonic acid (AMPS),hexametaphosphate salts, zinc citrate trihydrate, polypeptides,polyolefin sulfonates, polyolefin phosphates, diphosphonates. Theinvention thus may comprise phosphate salts. In particular embodiments,these salts are alkali phosphate salts, i.e., salts of alkali metalhydroxides or alkaline earth hydroxides, for example, sodium, potassiumor calcium salts. “Phosphate” as used herein encompasses orallyacceptable mono- and polyphosphates, for example, P1-6 phosphates, forexample monomeric phosphates such as monobasic, dibasic or tribasicphosphate; dimeric phosphates such as pyrophosphates; and multimericphosphates, e.g., sodium hexametaphosphate. In particular examples, theselected phosphate is selected from alkali dibasic phosphate and alkalipyrophosphate salts, e.g., selected from sodium phosphate dibasic,potassium phosphate dibasic, dicalcium phosphate dihydrate, calciumpyrophosphate, tetrasodium pyrophosphate, tetrapotassium pyrophosphate,sodium tripolyphosphate, and mixtures of any of two or more of these. Ina particular embodiment, for example the compositions comprise a mixtureof tetrasodium pyrophosphate (Na₄P₂O₇), calcium pyrophosphate (Ca₂P₂O₇),and sodium phosphate dibasic (Na₂HPO₄), e.g., in amounts of ca. 3-4% ofthe sodium phosphate dibasic and ca. 0.2-1% of each of thepyrophosphates. In another embodiment, the compositions comprise amixture of tetrasodium pyrophosphate (TSPP) and sodium tripolyphosphate(STPP)(Na₅P₃O₁₀), e.g., in proportions of TSPP at about 1-2% and STPP atabout 7% to about 10%. Such phosphates are provided in an amounteffective to reduce erosion of the enamel, to aid in cleaning the teeth,and/or to reduce tartar buildup on the teeth, for example in an amountof 2-20%, e.g., ca. 5-15%, by weight of the composition.

Polymers:

The oral care compositions of the invention may also include additionalpolymers to adjust the viscosity of the formulation or enhance thesolubility of other ingredients. Such additional polymers includepolyethylene glycols, polyvinyl methyl ether maleic acid copolymers,polysaccharides (e.g., cellulose derivatives, for example carboxymethylcellulose, or polysaccharide gums, for example xanthan gum orcarrageenan gum). Acidic polymers, for example polyacrylate gels, may beprovided in the form of their free acids or partially or fullyneutralized water soluble alkali metal (e.g., potassium and sodium) orammonium salts. Certain embodiments include 1:4 to 4:1 copolymers ofmaleic anhydride or acid with another polymerizable ethylenicallyunsaturated monomer, for example, methyl vinyl ether (methoxyethylene)having a molecular weight (M.W.) of about 30,000 to about 1,000,000.These copolymers are available for example as Gantrez AN 139(M.W.500,000), AN 1 19 (M.W. 250,000) and S-97 Pharmaceutical Grade (M.W.70,000), of GAF Chemicals Corporation.

Other operative polymers include those such as the 1:1 copolymers ofmaleic anhydride with ethyl acrylate, hydroxyethyl methacrylate,N-vinyl-2-pyrollidone, or ethylene, the latter being available forexample as Monsanto EMA No. 1 103, M.W. 10,000 and EMA Grade 61, and 1:1copolymers of acrylic acid with methyl or hydroxyethyl methacrylate,methyl or ethyl acrylate, isobutyl vinyl ether or N-vinyl-2-pyrrolidone.

Suitable generally, are polymerized olefinically or ethylenicallyunsaturated carboxylic acids containing an activated carbon-to-carbonolefinic double bond and at least one carboxyl group, that is, an acidcontaining an olefinic double bond which readily functions inpolymerization because of its presence in the monomer molecule either inthe alpha-beta position with respect to a carboxyl group or as part of aterminal methylene grouping. Illustrative of such acids are acrylic,methacrylic, ethacrylic, alpha-chloroacrylic, crotonic, beta-acryloxypropionic, sorbic, alpha-chlorsorbic, cinnamic, beta-styrylacrylic,muconic, itaconic, citraconic, mesaconic, glutaconic, aconitic,alpha-phenylacrylic, 2-benzyl acrylic, 2-cyclohexylacrylic, angelic,umbellic, fumaric, maleic acids and anhydrides. Other different olefinicmonomers copolymerizable with such carboxylic monomers includevinylacetate, vinyl chloride, dimethyl maleate and the like. Copolymerscontain sufficient carboxylic salt groups for water-solubility.

A further class of polymeric agents includes a composition containinghomopolymers of substituted acrylamides and/or homopolymers ofunsaturated sulfonic acids and salts thereof, in particular wherepolymers are based on unsaturated sulfonic acids selected fromacrylamidoalykane sulfonic acids such as 2-acrylamide 2 methylpropanesulfonic acid having a molecular weight of about 1,000 to about2,000,000, described in U.S. Pat. No. 4,842,847, Jun. 27, 1989 to Zahid,incorporated herein by reference.

Another useful class of polymeric agents includes polyamino acids,particularly those containing proportions of anionic surface-activeamino acids such as aspartic acid, glutamic acid and phosphoserine, asdisclosed in U.S. Pat. No. 4,866,161 Sikes et al., incorporated hereinby reference.

Silica thickeners, which form polymeric structures or gels in aqueousmedia, may be present. Note that these silica thickeners are physicallyand functionally distinct from the particulate silica abrasives alsopresent in the compositions, as the silica thickeners are very finelydivided and provide little or no abrasive action. Other thickeningagents are carboxyvinyl polymers, carrageenan, hydroxyethyl celluloseand water soluble salts of cellulose ethers such as sodium carboxymethylcellulose and sodium carboxymethyl hydroxyethyl cellulose. Natural gumssuch as karaya, gum arabic, and gum tragacanth can also be incorporated.Colloidal magnesium aluminum silicate can also be used as component ofthe thickening composition to further improve the composition's texture.In certain embodiments, thickening agents in an amount of about 0.5% toabout 5.0% by weight of the total composition are used.

The compositions of the invention may include an anionic polymer, forexample in an amount of from about 0.05 to about 5%. Such agents areknown generally for use in dentifrice, although not for this particularapplication, useful in the present invention are disclosed in U.S. Pat.Nos. 5,188,821 and 5,192,531; and include synthetic anionic polymericpolycarboxylates, such as 1:4 to 4:1 copolymers of maleic anhydride oracid with another polymerizable ethylenically unsaturated monomer,preferably methyl vinyl ether/maleic anhydride having a molecular weight(M.W.) of about 30,000 to about 1,000,000, most preferably about 300,000to about 800,000. These copolymers are available for example as Gantrez.e.g., AN 139 (M.W. 500,000), AN 119 (M.W. 250,000) and preferably S-97Pharmaceutical Grade (M.W. 700,000) available from ISP Technologies,Inc., Bound Brook, N.J. The enhancing agents when present are present inamounts ranging from about 0.05 to about 3% by weight. Other operativepolymers include those such as the 1:1 copolymers of maleic anhydridewith ethyl acrylate, hydroxyethyl methacrylate, N-vinyl-2-pyrollidone,or ethylene, the latter being available for example as Monsanto EMA No.1103, M.W. 10,000 and EMA Grade 61, and 1:1 copolymers of acrylic acidwith methyl or hydroxyethyl methacrylate, methyl or ethyl acrylate,isobutyl vinyl ether or N-vinyl-2-pyrrolidone. Suitable generally, arepolymerized olefinically or ethylenically unsaturated carboxylic acidscontaining an activated carbon-to-carbon olefinic double bond and atleast one carboxyl group, that is, an acid containing an olefinic doublebond which readily functions in polymerization because of its presencein the monomer molecule either in the alpha-beta position with respectto a carboxyl group or as part of a terminal methylene grouping.Illustrative of such acids are acrylic, methacrylic, ethacrylic,alpha-chloroacrylic, crotonic, beta-acryloxy propionic, sorbic,alpha-chlorsorbic, cinnamic, beta-styrylacrylic, muconic, itaconic,citraconic, mesaconic, glutaconic, aconitic, alpha-phenylacrylic,2-benzyl acrylic, 2-cyclohexylacrylic, angelic, umbellic, fumaric,maleic acids and anhydrides. Other different olefinic monomerscopolymerizable with such carboxylic monomers include vinylacetate,vinyl chloride, dimethyl maleate and the like. Copolymers containsufficient carboxylic salt groups for water-solubility. A further classof polymeric agents includes a composition containing homopolymers ofsubstituted acrylamides and/or homopolymers of unsaturated sulfonicacids and salts thereof, in particular where polymers are based onunsaturated sulfonic acids selected from acrylamidoalykane sulfonicacids such as 2-acrylamide 2 methylpropane sulfonic acid having amolecular weight of about 1,000 to about 2,000,000, described in U.S.Pat. No. 4,842,847, Jun. 27, 1989 to Zahid. Another useful class ofpolymeric agents includes polyamino acids containing proportions ofanionic surface-active amino acids such as aspartic acid, glutamic acidand phosphoserine, e.g. as disclosed in U.S. Pat. No. 4,866,161 Sikes etal.

Water:

The oral compositions may comprise significant levels of water. Wateremployed in the preparation of commercial oral compositions should bedeionized and free of organic impurities. The amount of water in thecompositions includes the free water which is added plus that amountwhich is introduced with other materials.

Humectants:

Within certain embodiments of the oral compositions, it is alsodesirable to incorporate a humectant to prevent the composition fromhardening upon exposure to air. Certain humectants can also impartdesirable sweetness or flavor to dentifrice compositions. Suitablehumectants include edible polyhydric alcohols such as glycerine,sorbitol, xylitol, propylene glycol as well as other polyols andmixtures of these humectants. In one embodiment of the invention, theprincipal humectant is glycerin, which may be present at levels ofgreater than 25%, e.g. 25-35% about 30%, with 5% or less of otherhumectants.

Other Optional Ingredients:

In addition to the above-described components, the embodiments of thisinvention can contain a variety of optional dentifrice ingredients someof which are described below. Optional ingredients include, for example,but are not limited to, adhesives, sudsing agents, flavoring agents,sweetening agents, additional antiplaque agents, abrasives, and coloringagents. These and other optional components are further described inU.S. Pat. No. 5,004,597, to Majeti; U.S. Pat. No. 3,959,458 to Agricolaet al. and U.S. Pat. No. 3,937,807, to Haefele, all being incorporatedherein by reference.

The composition can be any type of composition. In certain embodiments,the composition is any composition in which it is desired to include anantibacterial agent for application to the skin. Examples of suchcompositions include, but are not limited to, personal carecompositions, antiperspirants, deodorants, body washes, shower gels, barsoaps, shampoo, hair conditioners and cosmetics.

Basic Amino Acids:

The basic amino acids which can be used in the compositions and methodsof the invention include not only naturally occurring basic amino acids,such as arginine, lysine, and histidine, but also any basic amino acidshaving a carboxyl group and an amino group in the molecule, which arewater-soluble and provide an aqueous solution with a pH of 7 or greater.

Accordingly, basic amino acids include, but are not limited to,arginine, lysine, serine, citrullene, ornithine, creatine, histidine,diaminobutanoic acid, diaminoproprionic acid, salts thereof orcombinations thereof. In a particular embodiment, the basic amino acidsare selected from arginine, citrullene, and ornithine.

In certain embodiments, the basic amino acid is arginine, for example,L-arginine, or a salt thereof.

Suitable salts include salts known in the art to be pharmaceuticallyacceptable salts and are generally considered to be physiologicallyacceptable in the amounts and concentrations provided. Physiologicallyacceptable salts include those derived from pharmaceutically acceptableinorganic or organic acids or bases, for example acid addition saltsformed by acids which form a physiological acceptable anion, e.g.,hydrochloride or bromide salt, and base addition salts formed by baseswhich form a physiologically acceptable cation, for example thosederived from alkali metals such as potassium and sodium or alkalineearth metals such as calcium and magnesium. Physiologically acceptablesalts may be obtained using standard procedures known in the art, forexample, by reacting a sufficiently basic compound such as an amine witha suitable acid affording a physiologically acceptable anion.

In certain embodiments, the basic amino acid is present in an amountcorresponding to 0.1% to 15%, e.g., 0.1 weight % to 10 weight %, e.g.,0.1 to 5 wt %, e.g., 0.5 weight % to 3 weight % of the total compositionweight, about e.g., 1%, 1.5%, 2%, 3%, 4%, 5%, or 8%, wherein the weightof the basic amino acid is calculated as free form.

Surfactants:

The invention may, in some embodiments, contain anionic surfactants, forexample, water-soluble salts of higher fatty acid monoglyceridemonosulfates, such as the sodium salt of the monosulfated monoglycerideof hydrogenated coconut oil fatty acids such as sodium N-methyl N-cocoyltaurate, sodium cocomo-glyceride sulfate; higher alkyl sulfates, such assodium lauryl sulfate; higher alkyl-ether sulfates, e.g., of formulaCH₃(CH₂)_(m)CH₂(OCH₂CH₂)_(n)OSO₃X, wherein m is 6-16, e.g., 10, n is1-6, e.g., 2, 3 or 4, and X is Na or, for example sodium laureth-2sulfate (CH₃(CH₂)₁₀CH₂(OCH₂CH₂)₂OSO₃Na); higher alkyl aryl sulfonatessuch as sodium dodecyl benzene sulfonate (sodium lauryl benzenesulfonate); higher alkyl sulfoacetates, such as sodium laurylsulfoacetate (dodecyl sodium sulfoacetate), higher fatty acid esters of1,2 dihydroxy propane sulfonate, sulfocolaurate (N-2-ethyl lauratepotassium sulfoacetamide) and sodium lauryl sarcosinate. By “higheralkyl” is meant, e.g., C₆₋₃o alkyl. In particular embodiments, theanionic surfactant (where present) is selected from sodium laurylsulfate and sodium ether lauryl sulfate. When present, the anionicsurfactant is present in an amount which is effective, e.g., >0.001% byweight of the formulation, but not at a concentration which would beirritating to the oral tissue, e.g., 1%, and optimal concentrationsdepend on the particular formulation and the particular surfactant. Inone embodiment, the anionic surfactant is present at from 0.03% to 5% byweight, e.g., 1.5%.

In another embodiment, cationic surfactants useful in the presentinvention can be broadly defined as derivatives of aliphatic quaternaryammonium compounds having one long alkyl chain containing 8 to 18 carbonatoms such as lauryl trimethylammonium chloride, cetyl pyridiniumchloride, cetyl trimethylammonium bromide,di-isobutylphenoxyethyldimethylbenzylammonium chloride, coconutalkyltrimethylammonium nitrite, cetyl pyridinium fluoride, and mixturesthereof. Illustrative cationic surfactants are the quaternary ammoniumfluorides described in U.S. Pat. No. 3,535,421, to Briner et al., hereinincorporated by reference. Certain cationic surfactants can also act asgermicides in the compositions.

Illustrative nonionic surfactants that can be used in the compositionsof the invention can be broadly defined as compounds produced bycondensation of alkylene oxide groups (hydrophilic in nature) with anorganic hydrophobic compound which may be aliphatic or alkylaromatic innature. Examples of suitable nonionic surfactants include, but are notlimited to, the Pluronics, polyethylene oxide condensates of alkylphenols, products derived from the condensation of ethylene oxide withthe reaction product of propylene oxide and ethylene diamine, ethyleneoxide condensates of aliphatic alcohols, long chain tertiary amineoxides, long chain tertiary phosphine oxides, long chain dialkylsulfoxides and mixtures of such materials. In a particular embodiment,the composition of the invention comprises a nonionic surfactantselected from polaxamers (e.g., polaxamer 407), polysorbates (e.g.,polysorbate 20), polyoxyl hydrogenated castor oils (e.g., polyoxyl 40hydrogenated castor oil), and mixtures thereof.

Illustrative amphoteric surfactants of that can be used in thecompositions of the invention include betaines (such ascocamidopropylbetaine), derivatives of aliphatic secondary and tertiaryamines in which the aliphatic radical can be a straight or branchedchain and wherein one of the aliphatic substituents contains about 8-18carbon atoms and one contains an anionic water-solubilizing group (suchas carboxylate, sulfonate, sulfate, phosphate or phosphonate), andmixtures of such materials.

The surfactant or mixtures of compatible surfactants can be present inthe compositions of the present invention in 0.1% to 5%, in anotherembodiment 0.3% to 3% and in another embodiment 0.5% to 2% by weight ofthe total composition.

Flavoring Agents:

The oral care compositions of the invention may also include a flavoringagent. Flavoring agents which are used in the practice of the presentinvention include, but are not limited to, essential oils and variousflavoring aldehydes, esters, alcohols, and similar materials, as well assweeteners such as sodium saccharin. Examples of the essential oilsinclude oils of spearmint, peppermint, wintergreen, sassafras, clove,sage, eucalyptus, marjoram, cinnamon, lemon, lime, grapefruit, andorange. Also useful are such chemicals as menthol, carvone, andanethole. Certain embodiments employ the oils of peppermint andspearmint.

The flavoring agent is incorporated in the oral composition at aconcentration of 0.01 to 1.5% by weight.

Chelating and Anti-Calculus Agents:

The oral care compositions of the invention also may include one or morechelating agents able to complex calcium found in the cell walls of thebacteria. Binding of this calcium weakens the bacterial cell wall andaugments bacterial lysis.

Another group of agents suitable for use as chelating or anti-calculusagents in the present invention are the soluble pyrophosphates. Thepyrophosphate salts used in the present compositions can be any of thealkali metal pyrophosphate salts. In certain embodiments, salts includetetra alkali metal pyrophosphate, dialkali metal diacid pyrophosphate,trialkali metal monoacid pyrophosphate and mixtures thereof, wherein thealkali metals are sodium or potassium. The salts are useful in boththeir hydrated and unhydrated forms. An effective amount ofpyrophosphate salt useful in the present composition is generally enoughto provide least 0.1 weight % pyrophosphate ions, e.g., 0.1 to 3 wt 5,e.g., 0.1 to 2 weight %, e.g., 0.1 to 1 wt %, e.g., 0.2 to 0.5 wt %. Thepyrophosphates also contribute to preservation of the compositions bylowering water activity.

In preparing oral care compositions, it is sometimes necessary to addsome thickening material to provide a desirable consistency or tostabilize or enhance the performance of the formulation. In certainembodiments, the thickening agents are carboxyvinyl polymers,carrageenan, xanthan gum, hydroxyethyl cellulose and water soluble saltsof cellulose ethers such as sodium carboxymethyl cellulose and sodiumcarboxymethyl hydroxyethyl cellulose. Natural gums such as karaya, gumarabic, and gum tragacanth can also be incorporated. Silica may also beavailable as a thickening agent, e.g., synthetic amorphous silica.Colloidal magnesium aluminum silicate or finely divided silica can beused as component of the thickening composition to further improve thecomposition's texture. In certain embodiments, thickening agents in anamount of about 0.5% to about 5.0% by weight of the total compositionare used. Thickeners may be present in an amount of from 1 weight % to15 weight %, from 3 weight % to 10 weight %, 4 weight % to 9 weight %,from 5 weight % to 8 weight %, for example 5 weight %, 6 weight %, 7weight %, or 8 weight %.

Abrasives:

Natural calcium carbonate is found in rocks such as chalk, limestone,marble and travertine. It is also the principle component of egg shellsand the shells of mollusks. The natural calcium carbonate abrasive ofthe invention is typically a finely ground limestone which mayoptionally be refined or partially refined to remove impurities. For usein the present invention, the material has an average particle size ofless than 10 microns, e.g., 3-7 microns, e.g. about 5.5 microns. Forexample, a small particle silica may have an average particle size (D50)of 2.5-4.5 microns. Simply because natural calcium carbonate may containa high proportion of relatively large particles of not carefullycontrolled, which may unacceptably increase the abrasivity, preferablyno more than 0.01%, preferably no more than 0.004% by weight ofparticles would not pass through a 325 mesh. The material has strongcrystal structure, and is thus much harder and more abrasive thanprecipitated calcium carbonate. The tap density for the natural calciumcarbonate is for example between 1 and 1.5 g/cc, e.g., about 1.2 forexample about 1.19 g/cc. There are different polymorphs of naturalcalcium carbonate, e.g., calcite, aragonite and vaterite, calcite beingpreferred for purposes of this invention. An example of a commerciallyavailable product suitable for use in the present invention includesVicron® 25-11 FG from GMZ.

Precipitated calcium carbonate is generally made by calcining limestone,to make calcium oxide (lime), which can then be converted back tocalcium carbonate by reaction with carbon dioxide in water. Precipitatedcalcium carbonate has a different crystal structure from natural calciumcarbonate. It is generally more friable and more porous, thus havinglower abrasivity and higher water absorption. For use in the presentinvention, the particles are small, e.g., having an average particlesize of 1-5 microns, and e.g., no more than 0.1%, preferably no morethan 0.05% by weight of particles which would not pass through a 325mesh. The particles may for example have a D50 of 3-6 microns, forexample 3.8=4.9, e.g., about 4.3; a D50 of 1-4 microns, e.g. 2.2-2.6microns, e.g., about 2.4 microns, and a D10 of 1-2 microns, e.g.,1.2-1.4, e.g. about 1.3 microns. The particles have relatively highwater absorption, e.g., at least 25 g/100 g, e.g. 30-70 g/100 g.Examples of commercially available products suitable for use in thepresent invention include, for example, Carbolag® 15 Plus from LagosIndustria Quimica.

In certain embodiments the invention may comprise additionalcalcium-containing abrasives, for example calcium phosphate abrasive,e.g., tricalcium phosphate (Ca₃(PO₄)₂), hydroxyapatite(Ca₁₀(PO₄)₆(OH)₂), or dicalcium phosphate dihydrate (CaHPO₄.2H₂O, alsosometimes referred to herein as DiCal) or calcium pyrophosphate, and/orsilica abrasives, sodium metaphosphate, potassium metaphosphate,aluminum silicate, calcined alumina, bentonite or other siliceousmaterials, or combinations thereof.

In certain embodiments, any silica suitable for oral care compositionsmay be used, such as precipitated silicas or silica gels. For example,the silica can also be small particle silica (e.g., Sorbosil AC43 fromPQ, Warrington, United Kingdom). The composition preferable containsfrom 5 to 20 weight % small particle silica, or for example 10-15 weight%, or for example 5 weight %, 10 wt %, 15 weight % or 20 weight % smallparticle silica.

In another embodiment, the abrasive may be high cleaning precipitatedsilica having a pellicle cleaning ratio (PCR) of greater than 85 whentested at 20% loading is known in the art as high cleaning silica.Typically, high cleaning silica also has a mean particle size d₅₀ offrom 5 to 15 μm and an oil absorption of from 40 to 120 cm³/100 gsilica. The cleaning efficacy of the precipitated silica is expressedusing the pellicle cleaning ratio (PCR). This is typically measured at20% silica loading. The high cleaning silica preferably has a PCR valueof greater than 85. The efficacy of the precipitated silica can also beexpressed with reference to its abrasive characteristic using theradioactive dentin abrasion (RDA). Ideally, RDA values for an oralcomposition should be below about 250 to protect tooth enamel/dentin.Methods of performing PCR and RDA are described in e.g., U.S. Pat. Nos.5,939,051 and 6,290,933 and “In Vitro Removal of Stain With Dentifrice”,G. K. Stookey et al., J. Dental Research, Vol. 61, pages 1236-9,November 1982. Typically, the precipitated silica has a mean particlesize d₅₀ of from 5 to 15 μm and an oil absorption of from 40 to 120cm³/100 g silica. Examples of precipitated silica having a mean particlesize d₅₀ of from 5 to 15 μm and an oil absorption of from 40 to 120cm³/100 g silica including commercially available silicas such asZeodent® 103 and Zeodent® 105 (Huber Silica Americas).

The composition preferable contains from 3 to 20 weight % high cleaningprecipitated silica, or for example 10-15 weight %, or for example 5weight %, 10 wt %, 15 weight % or 20 weight % high cleaning precipitatedsilica.

The composition may also comprise an abrasive silica having an acid pHin the composition. For example, prophy silica available from Grace,offered as Sylodent™, can be used. The acidic silica abrasive isincluded in the dentifrice components at a concentration of about 2 toabout 35% by weight; about 3 to about 20% by weight, about 3 to about15% by weight, about 10 to about 15% by weight. In certain embodiments,the acidic silica abrasive may be present in an amount between 2-7%. Inother embodiments, it may be present in an amount between 7-15% byweight. Still on other embodiments, it may be present in an amountbetween 15-30% by weight. For example, the acidic silica abrasive may bepresent in an amount selected from 2 wt. %, 3 wt. %, 4% wt. %, 5 wt. %,6 wt. %, 7 wt. %, 8 wt. %, 9 wt. %, 10 wt. %, 11 wt. %, 12 wt. %, 13 wt.%, 14 wt. %, 15 wt. %, 16 wt. %, 17 wt. %, 18 wt. %, 19 wt. %, 20 wt. %.

A commercially available acidic silica abrasive is Sylodent 783available from W. R. Grace & Company (Baltimore, Md.). Sylodent 783 hasa pH of 3.4-4.2 when measured as a 5% by weight slurry in water. For usein the present invention, the silica material has an average particlesize of less than 10 microns, e.g., 3-7 microns, e.g. about 5.5 microns.

In some embodiments, the compositions of the present disclosure containa buffering agent. Examples of buffering agents include anhydrouscarbonates such as sodium carbonate, sesquicarbonates, bicarbonates suchas sodium bicarbonate, silicates, bisulfates, phosphates (e.g.,monopotassium phosphate, dipotassium phosphate, tribasic sodiumphosphate, sodium tripolyphosphate, phosphoric acid), citrates (e.g.citric acid, trisodium citrate dehydrate), pyrophosphates (sodium andpotassium salts) and combinations thereof. The amount of buffering agentis sufficient to provide a pH of about 5 to about 9, preferable about 6to about 8, and more preferable about 7, when the composition isdissolved in water, a mouthrinse base, or a toothpaste base. Typicalamounts of buffering agent are about 5% to about 35%, in one embodimentabout 10% to about 30%, in another embodiment about 15% to about 25%, byweight of the total composition.

Various other materials may be incorporated in the oral preparations ofthis invention such as whitening agents, preservatives, silicones,chlorophyll compounds and/or ammoniated material such as urea,diammonium phosphate, and mixtures thereof. These adjuvants, wherepresent, are incorporated in the preparations in amounts which do notsubstantially adversely affect the properties and characteristicsdesired.

Any suitable flavoring or sweetening material may also be employed.Examples of suitable flavoring constituents are flavoring oils, e.g. oilof spearmint, peppermint, wintergreen, sassafras, clove, sage,eucalyptus, marjoram, cinnamon, lemon, and orange, and methylsalicylate. Suitable sweetening agents include sucrose, lactose,maltose, xylitol, sodium cyclamate, perillartine, AMP (aspartyl phenylalanine, methyl ester), saccharine and the like. Suitably, flavor andsweetening agents may each or together comprise from about 0.1% to 5%more of the preparation. Moreover, flavoring oil is believed to aid thedissolving of the antibacterial agent, together with or even in theabsence of surface-active agent.

The present invention in its method aspect involves applying to the oralcavity a safe and effective amount of the compositions described herein.

The following examples further describe and demonstrate illustrativeembodiments within the scope of the present invention. The examples aregiven solely for illustration and are not to be construed as limitationsof this invention as many variations are possible without departing fromthe spirit and scope thereof. Various modifications of the invention inaddition to those shown and described herein should be apparent to thoseskilled in the art and are intended to fall within the appended claims.

Methods and Materials:

Naringin was obtained from Perfect Health, LLC (Lot CYPD-A-01468, 99%HPLC purity, Somerset, N.J.) or Sigma-Aldrich (St. Louis, Mo.) cat#71162 or N1376. Zinc (II) Acetate salt was obtained from Sigma-Aldrich(St. Louis, Mo.) cat #383317.

Example 1—Synthesis Method 1

Briefly, 1.25e-4 moles of Naringin (ligand) solution was prepared in 10ml of methanol (J. T. Baker, cat #9093-03). Next, a 2 ml solution ofZinc (II) Acetate (1.25e-4 moles) in distilled water was slowly addedwith constant stirring. The pH of the solution was adjusted to around10.0 using sodium hydroxide (50% solution) (J.T. Baker, Center Valley,Pa., cat #9721-02). The reaction was allowed to continue for around 4hours at room temperature. The precipitate was filtered in a vacuumsystem, washed with water and then air dried. The naringin:Zinc complex,observed as a yellowish colored precipitate, was obtained, characterizedusing UV-VIS, FTIR and ¹H NMR and labeled as Product 1.

Example 2—Synthesis Method 2

Firstly, 1.25e-4 moles of Naringin (ligand) solution was prepared in 10ml of double distilled H₂O and heated to around 70° C. to enhance thesolubility of Naringin. The resultant solution was then filtered. Next,2 ml of Zinc (II) Acetate (1.25e-4 moles) in double distilled water wasslowly added under constant stirring. The pH of the solution was thenadjusted to around 10.0 using sodium hydroxide (50% solution). Thereaction was allowed to incubate for around 2 hours. The formedprecipitate was collected, washed initially with dDH₂O followed by 200proof Ethanol. The precipitate, observed as yellowish colored, was thenvacuum dried, collected, characterized using UV-Vis spectroscopy, FTIRand ¹H-NMR and labeled as Product 2.

Example 3—Synthesis Method 3

Briefly, 1.25e-4 moles of Naringin (ligand) solution was prepared in 10ml of double distilled H₂O and heated to around 70° C. to enhance thesolubility of Naringin. The pH of the solution was adjusted to around10.0 using sodium hydroxide (50% solution). Next, 2 ml of Zinc (II)Acetate (1.25e-4 moles) in double distilled water was slowly added underconstant stirring. The precipitate formed, observed as yellowishcolored, was collected, washed initially with dDH₂O followed by 200proof Ethanol, vacuum dried, characterized using UV-Vis spectroscopy,FTIR and ¹H-NMR and labeled as Product 3.

Example 4—Synthesis Method 4

Either 1.25e-4 moles or 2.50e-4 moles of Naringin (ligand) solution wasprepared in 10 ml of double distilled H₂O and heated to around 70° C. toenhance the solubility of Naringin. The resultant solution was filtered.Next, 2 ml solution of Zinc (II) Acetate (1.25e-4 moles) in doubledistilled water was slowly added under constant stirring. The pH of thesolution was then adjusted to around 7.0 using sodium hydroxide (50%solution). The precipitate formed was collected, washed initially withdDH₂O followed by 200 proof ethanol. The resulting precipitate, observedas yellowish colored, was vacuum dried, collected, characterized usingUV-Vis spectroscopy, FTIR and ¹H-NMR and labeled as Product 4 (1.25e-4moles Naringin) and 5 (2.50e-4 moles Naringin).

Example 5—Synthesis Method 5

To produce a Product 6, 2.50e-4 moles of Naringin (ligand) solution wasprepared in 10 ml of 7% Propylene Glycol solution pre-warmed to around70° C. Without being bound to theory, it is believed that thistemperature enhances the solubility of Naringin. The mixture (slightlycloudy and yellowish in color) was constantly stirred over a heatedstirring plate maintained at 45° C. In certain embodiments, thetemperature range can be 40-50° C. The pH of the solution was adjustedusing sodium hydroxide until a clear orange color solution was obtained(pH 9-10). Next, 2 ml of Zinc (II) Acetate (1.25e-4 moles) in 7%Propylene Glycol solution, pre-warmed to around 70° C., was slowly addedwith constant stirring. The reaction was incubated for approximately 2hours. A precipitate formed, was collected, washed initially with dDH₂O,then with 200 proof ethanol.

Example 6—Characterization with UV-Vis

The UV-Vis spectra of naringin:Zinc complexes were generated using aSpectraMax M5 Plate Reader. Briefly, 2.0% samples of naringin:Zinccomplexes or Naringin starting material were prepared in 100% DMSO (BDH,cat# BDH 1115-1LP). Samples were then subjected to a 1:1 serial dilutionin a 96 well clear flat bottom plate such that the highest concentrationwas 1.0% and the lowest concentration 0.0078% in 100 ul of DMSO. Sampleswere subjected to spectra scan from 200 nm wavelength to 500 nmwavelength. Plots of optical density (OD) vs wavelength for all serialdilutions were determined but only the results from 0.03125% dilutionwere presented in the Results section.

As shown in FIGS. 2-4, free Naringin exhibits an absorption maximum peakin aqueous solution at 290 nm, corresponding to the A ring absorption(benzoyl system), and a weaker band at 325 nm, corresponding to the Bring absorption (cinnamoyl system) see Malesev et al., J. Serb. Chem.Soc., 2007, 72(10): 921-39. Upon binding with Zinc Acetate to form thecomplex, a shift in the weaker absorption band from 325 nm to 365 nm wasnoted for Product 1. As shown in FIG. 3, Product 2 produced a subtleshift at the maximum peak from 290 nm to 295 nm. A more dramatic shiftwas also observed for the weaker band from 325 nm to 370 nm. Product 3provided a similar profile. As shown in FIG. 4, Products 4 and 5 bothexhibited similar profiles. A shift in the maximal peak (290 nm to 295nm) and the weaker band (325 nm to 370 nm) was observed suggesting aninteraction of the Zinc (II) ion with the condensed ring of theflavanone. The shift was previously attributed to an increasedconjugative effect resulting from new ring formation upon metalcomplexation (Malesev et al., supra). This result is consistent with thefindings from FTIR and ¹H-NMR studies (see FIG. 5-FIG. 7B). The maximalpeak shift was seen on most occasions for the various products tested,but not during every test. See, for example, Product 1 compared toProduct 2 in Table 1.

TABLE 1 Summary showing the peak position in A-ring absorption andB-ring absorption for free Naringin and naringin:Zinc complexessynthesized under various conditions. Samples Maximal Peak Weaker BandNaringin (free) 290 nm 325 nm Product 1 290 nm 365 nm Product 2 295 nm370 nm Product 3 295 nm 370 nm Product 4 295 nm 370 nm Product 5 295 nm370 nm

Example 7—Characterization with FTIR

Infrared spectra were collected using a Bruker Vertex 70 FTIRspectrometer equipped with a GladiATR diamond ATR accessory (Piketechnologies, Madison, Wis.). The spectral range was 80-4000 cm⁻¹ and aresolution of 4 cm⁻¹ was used. All measurements were carried out onpowdered samples at room temperature.

FIG. 5 displays the infrared spectra of free Naringin and Product 4complex. The comparison of spectral data reveals pronounced differencesin the vibrational response of Naringin upon interaction with Zinc. Asan example, the infrared spectrum of Naringin exhibits a strong bandnear 1645 cm⁻¹ which is assigned to the stretching vibration of thecarbonyl group υ(C═O) (Pereira et al., Molecules, 2007, 12: 1352-66; Liet al., Spectrochimica Acta Part A, 2007, 67:395-401; Yousuf et al.,International Journal of Spectroscopy, 2014, Article ID 562160). In thepresence of Zinc this band is red-shifted to 1615 cm⁻¹ suggestingcoordination of Naringin to Zinc via the carbonyl oxygen. Similarbehavior has been previously observed for other metal-flavonoidcomplexes (Pereira et al., Li et al., Yousuf et al., supra). In additionto the carbonyl moiety, the 5-OH group was found to be another importantsite for metal chelation in 5-hydroxyflavone derivatives. For theProduct 4 complex, bands associated with υ(C—O(H)) (or C—C—O) stretchingvibrations of phenolic group are coupled with δ(OH) deformation modesand occur in the 1230-1370 cm⁻¹ range. As can be seen from FIG. 5, thesebands display pronounced frequency shifts and relative intensity changesin comparison with the free naringin. This behavior together with theenergy shift of the υ(C═O) band at 1645 cm⁻¹, suggest that zinccomplexation with naringin likely takes place through the oxygen of thecarbonyl group and the 5-hydroxyl group. Additional peak shifts andintensity variations in the bands of aromatic and disaccharide units ofProduct 4 may arise from the redistribution of electrons on the rings,as a result of a new ring formation, and conformational changes inglucoside, respectively. Finally, an additional band observed near 460cm⁻¹ in complex Product 4 may originate from the υ (Zn—O) stretchingvibration (Yousuf et al., supra) further supporting naringin:Zinccomplexation.

FIG. 6 compares the infrared response of naringin and naringin:zinccomplexes of Product 2, Product 4 and Product 5, which were preparedunder different pH and molar ratio conditions. Aside from differences intheir relative intensities, all compounds show an overall similarspectral profile and peak shifts suggesting their similar structuralfeatures.

Example 8—Characterization with ¹H NMR

The NMR measurements were performed on 10 wt % Naringin or naringin:Zincsamples in DMSO-d5 solutions. All ¹H NMR spectra were acquired on aBruker Avance 500 spectrometer working at 500 MHz for ¹H at roomtemperature. Diffusion coefficients of the naringin and naringin:Zncomplexes in DMSO-d5 were measured by ¹H Pulse-field gradient NMRspectroscopy at 25° C. All chemical shifts were reported in δ (ppm)using TMS as the internal standard.

The ¹H chemical shifts for Naringin and naringin:Zinc complexes arepresented in FIG. 7B and Table 2. Notably, the peak at 12 ppmcorresponding to 5-OH phenolic group is absent upon naringin:Zinccomplex formation, suggesting that this site conjugates with Zinc. Thepeaks assigned to be H6 and H8 of the A ring shift to lower frequencywhen forming complex. Without being bound to theory, this could bebecause the coordination increases the planarity of the flavonoidmolecules and/or because coordination induces the large conformationalchange of the disaccharide, thereby increasing the electron densities onthe ring A to shield proton H6 and H8.

TABLE 2 ¹H NMR chemical shifts (ppm) for Naringin and Naringin:Zinccomplexes. Sample Naringin Product 4 Product 5 Naringin:Zn 1:1 2:1 5-OH12.07  4′-OH 9.62 9.56 9.57 H2′, H6′ 7.34 (d, J = 8 Hz) 7.31 (d, J = 8Hz) 7.31 (d, J = 8 Hz) H3′, H5′ 6.80 (d, J = 8 Hz) 6.79 (d, J = 8 Hz)6.78 (d, J = 8 Hz) H8 6.18 5.63 5.63 H6 6.22 5.78 5.79 H3 3.17 3.20 3.18

Surprisingly, the ¹H NMR spectra as well as the FTIR spectra show thatthe naringin:zinc complex samples prepared from different ratios ofnaringin and zinc (2:1 or 1:1) were similar.

Example 9—Characterization with Diffusion Measurements

Diffusion measurements were performed using Bruker Avance 500spectrometer equipped with an observed broadband probe with a z axisgradient coil with maximum gradient strength of 72 G/cm. A doublestimulated echo pulse sequence with bipolar gradient pulses and twospoil gradients were used. The diffusion time was 0.1 second. Theduration of the field gradient pulse was adjusted to be 4 milliseconds.The pulse gradients were incremented from 5 to 95% of the maximumgradient strength in a linear ramp with a total of experimental time of45 minutes.

The molecular diffusion of Naringin:Zinc complex was performed to detectdifferences between samples prepared from 2:1 versus 1:1 Naringin:Zincmolar ratios. Without being bound to theory, it was postulated thatmolecular diffusion of a 2:1 should be slower than a 1:1 complex. Theobserved diffusion coefficients of the Naringin and Naringin:Zinccomplexes of Product 4 and Product 5 are summarized in FIG. 8. Thediffusion coefficients of the complexes are about 25% slower than thatof Naringin. It was also observed that the diffusion coefficients forall two Naringin:Zinc complexes tested were similar, regardless whetherthey were prepared from the initial mixing of 1:1 or 2:1 Naringin:Zincmolar ratios. These findings imply that both Naringin:Zinc complexeshave the same stoichiometric ratio of Naringin and Zinc.

The calculated diffusion coefficient was used to elucidate the size ofthe molecular species using the Stokes-Einstein relation:

$D = \frac{k_{B}T}{6\pi\;\eta\; r}$

where r is the hydrodynamic radius of a hard sphere moving in acontinuum fluid of viscosity η at temperature T and k_(B) is theBoltzmann constant. Assuming the diffusing species to be spherical, therelation of D and molecular weight MW can be described as

$D = {\frac{k_{B}T}{6\pi\;\eta\; r}\sqrt[3]{\frac{4\pi\;\rho\; N_{A}}{3{MW}}}}$

where p is the effective molecular density of the molecule and N_(A) isAvogadro's number. Assuming the Naringin and Naringin:Zinc complexesstudied herein have the same packing effect and geometry, the molecularweight of Naringin:Zinc complex can be estimated according to the knownmolecular weight of Naringin and diffusion coefficient D according tothe relationship:MW∝1/D ³

TABLE 3 Summary of the estimated molecular weight and stoichiometricratio of Naringin to Zinc based on diffusion NMR measurement. EstimatedEstimated Diffusion coefficient molecular weight stoichiometric ratioSample (×10⁻¹¹ m²/s) (mol/g) of Naringin to Zinc Naringin 3.99 ± 0.09580.54 — Product 5 3.11 ± 0.08 1228.4 2.1:1 Product 4 2.99 ± 0.08 1376.32.4:1

From Table 3, the estimated stoichiometric ratio of Naringin and Zincbased upon the estimated molecular weight was found to be approximately2:1 (Naringin:Zinc).

Example 10—Characterization of Melting Point

Melting points were recorded on an Electrothermal MEL-TEMP 3.0(Barnstead International, Dubuque, Iowa) melting point apparatus. Smallamounts of the samples were introduced into the capillary tube. The tubewas inserted into the holder and the samples were monitored from theobservation window for physical changes. Temperatures were recorded whenchanges occurred. Melting points for the samples are summarized in Table4. All samples showed disintegration in the range of 230-254.3° C.

TABLE 4 Summary of the melting points for naringin and naringin:zinccomplexes synthesized from methods described herein. Sample DescriptionTest Method Result Comments 1 Naringin melting point/range USP/EP165.5-168.1 turns light brown 2 Product 1 melting point/range USP/EP Nomelting color change observed: @239.7° C. observed up to turned brown380.9° C. 3 Product 2 melting point/range USP/EP No melting @230.0°C.-turned brown, @ observed up to 232.1° C. black hue notice in tube380.9° C. on top of sample. @254.3° C. sample turned black. 4 Product 3melting point/range USP/EP No melting @226.3° C. turned brown. Shortlyobserved up to after a black hue was notice in 380.9° C. tube on top ofsample. @248.4° C. sample turned black. 5 Product 4 melting point/rangeUSP/EP no melting 234.6° C. turned dark brown and observed started toshrink into a ball but did not liquify. By 239.4° C. a little bubblingwas noticed and eventually started to darken in color. 6 Product 5melting point/range USP/EP No melting @221.7° C. turned amber in color.observed up to @228.6° C. the sample seemed to 380.9° C. have “shrunk”or had contracted into a round-like form. @230.0° C. sample expandedupwards in the tube. Black hue was noticed in tube on top of sample.@254.3° C. sample turned black. 7 Product 6 melting point/range USP/EPNo melting Sample turned from yellow observed up to (initial color) todark orange @ 250.0° C. 225.1° C. Sample turned dark brown and it'sphysical form looked more compacted (shrunk in size) @ 228.0° C.Compacted sample darkened until practically black in color up to 250.0°C.

Example 11—Stability Characterization

To initiate stability testing of the complex, selective samples weredissolved in DMSO at 2% stock solution for initial UV—Visual spectra andallowed to sit for 6-8 weeks at room temperature. Samples were subjectedto 1:1 serial dilutions in DMSO and read using the SpectraMax M5 PlateReader. Results are summarized in FIG. 9 and FIG. 10. The shiftsobserved previously for the maximal peak (290 nm to 295 nm) and theweaker band (325 nm to 370 nm) were maintained 6-8 weeks post samplepreparation.

Example 12—Characterization of Antimicrobial Activity: Zone ofInhibition

Antimicrobial activity was determined using a zone of inhibition typeanalysis in combination with growth inhibition/toxicity testing. Forzone of inhibition determination, a single colony of Aggregatibacteractinomycetemcomitans (A.a) was cultured in approximately 30 ml of BHIbroth (BD, cat #237500) supplemented with 0.5% Yeast Extract (BD, cat#210941) in 37° C. and 5% CO₂ overnight. A 1:10 dilution of theovernight culture at (OD₆₁₀ 0.5) was prepared and a 100 μl culture wasaseptically transferred onto room temperature equilibrated TSAII(Trypticase soy agar with 5% sheep blood) plates (BD, cat #221239) andgently spread out using a sterile spreader. A sterile 6 mm diameterfilter disc was place on top of the cultured plate using sterile forcepsand 5 μl of a 100 mM test compound in DMSO was applied to the center ofthe filter disc along with the appropriate controls 1% chlorohexidine(CHX), 3% Triclosan, media only and DMSO. Treated plates were incubatedin 37° C., 5% CO₂ for 48 hours prior to observations and diameter ofzone of inhibition being measured. Table 5 summarizes the activity ofthe naringin:Zinc complex on the oral pathogen A.a.

TABLE 5 Antimicrobial effects of free Naringin vs. Naringin:Zinccomplexes represented by the formation of zone of inhibition aroundfilter discs. Zone of Inhibition in duplicates (diameter including thedisc itself) against Aggregatibacter actinomycetemcomitans) Samples (@500 nmoles) # 1 (cm) # 2 (cm) Naringin alone 0.6 × 0.6 0.6 × 0.6 (sizeof filter disc) (size of filter disc) Product 1 1.5 × 1.5 1.8 × 1.5Product 2 1.8 × 1.4 1.9 × 1.8 Product 3 1.1 × 1.2 1.2 × 1.4 Product 41.5 × 1.2 1.5 × 1.3 Product 5 ND ND

The zone of inhibition method yields qualitative results regarding theantimicrobial activity of the complexes. Appearance of zone ofinhibition around the disc is an indicative of antimicrobial activity ofthe test compound spotted onto the filter disc. The oral pathogen A.actinomycetemcomitans was chosen here because it is regarded as a keybacterial agent associated with aggressive periodontitis in young adultsand is implicated in adult forms of destructive periodontal disease(Darby et al., Periodontology 2000, 2001, 26:33-53). Data from the zoneof inhibition studies indicated that at 500 nmoles, the Naringin:Zinccomplex showed an increased potency in killing A.a compared to Naringinalone. DMSO alone did not show any killing while 1% Chlorohexidine did.

Example 13—Characterization of Antimicrobial Activity: GrowthInhibition/IC₅₀ Determination

Single species of Streptococcus gordonii (S. gordonii, S.g) strain V288(ATCC #35105), Streptococcus sobrinus (S. sobrinus, S.$) strain SL1[CCM6070, CNCTC 9/89] (ATCC #33478), or Aggregatibacteractinomycetemcomitans were analyzed. Also, a mixed culture containingActinomyces viscosus (A. viscosus, A.v) strain (ATCC, #43146),Lactobacillus casei (L. casei, L.c) strain (ATCC #334), Streptococcusoralis (S. oralis, S. o) strain (ATCC #35037), Fusobacterium nucleatum(F. nucleatum, F. n) strain (ATCC #10953), and Veilonella parvula (V.parvula, V. p) strain (ATCC #17745) was also analyzed for growthinhibition/toxicity activity. Bacteria were cultured overnight inapproximately 20 ml of BHI broth (BD, cat #237500) or maintained in aspecialized complex modified BHI II medium in a continuous culturechemostat at 37° C. respectively. The following day, the OD₆₁₀ weredetermined and solutions diluted to an OD₆₁₀ of 0.2 or 0.1 respectivelyfor the assay.

Test samples (naringin, naringin:Zinc complex and naringin+Zinc acetatecombination) were prepared as a 200 mM stock solution followed by 2 foldserial dilutions in 100% DMSO. Triclosan (positive control) was preparedas a 10% stock in 100% EtOH and 3 fold serial dilutions were performedin 100% EtOH. An appropriate amount of the diluted samples was thentransferred into a 96 well clear plate such that the resulting finalconcentration of the test samples ranged from either a 2 mM or 4 mM(highest concentration) to 0.0078 mM or 0.0156 mM (lowest concentrationrespectively) in 2% DMSO in 100 μl media per well. Triclosan (TCN)ranged from 0.01% to 1.67e-6% in 1% EtOH. For Zinc acetate, in order tomimic the 2:1 molar ratio (ligand:Zinc) in the Naringin:Zinc complex,the final concentration ranged from 0.7 mM to 0.0027 mM for samples with2 mM as the highest concentration and 1.33 mM to 0.052 mM for sampleswith 4 mM as the highest concentration in 2% DMSO. Next, 100 μl ofbacterial culture at OD₆₁₀ 0.2 were introduced to the wells with thetest samples. The wells were thoroughly mixed and incubated at 37° C.overnight. The plates were read using an Envision plate reader(PerkinElmer, Waltham, Mass.) at 610 nm. Representative findings aresummarized in FIG. 11.

The IC₅₀ for the complex, Product 5, was determined for Streptococcusgordonii (S. gordonii), Streptococcus sobrinus (S. sobrinus),Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), andalso for the mixed bacterial culture. The dose response curves wereplotted using GraphPad Prism 6 and representative plots are presented inFIGS. 11 and 22. Determined IC₅₀ values are summarized in Table 6. TheIC₅₀ values show that the complex was more potent than both the ZincAcetate alone as well as the combination of Naringin+Zinc Acetate takentogether. Triclosan (TCN) was included as a positive control forbacterial inhibition.

TABLE 6 Summary of IC₅₀ values for single and mixed cultures. IC 50 (mM)Combination of Naringin/Zinc Zinc Naringin Acetate Acetate IC50 (mM)Bacterial Strain alone Product 5 (2:1) alone TCN Streptococcus sobrinusN/A 0.22 ± 0.06 1.03 ± 0.57 0.29 ± 0.04 0.0060 ± 0.0017 Streptococcusgordonii N/A 0.22 ± 0.07  1.1 ± 0.15  0.3 ± 0.02  0.0060 ± 0.00085Aggregatibacter N/A 0.37 ± 0.18 2.10 ± 0.28 1.73 ± 0.95 0.00061 ±0.00046 actinomycetemcomitans Mixed Species (aerobic) N/A 0.1 0.67Ambiguous 0.0076 Mixed Species N/A 0.12 1.0 0.25 0.0048 (anaerobic)

The complex exhibited anti-microbial effects on cariogenic pathogen S.sobinus, commensal pathogen S.gordonii and pathogenic late colonizeroral microflora A. actinomycetemcomitans. These findings were extendedto mixed chemostat bacterial cultures as well. The concentration thatinhibited 50% of the bacterial growth ranged from 0.1-0.37 mM(corresponding to 122.6-453.5 ppm). Naringin in combination with ZincAcetate generated IC₅₀ values ranging from 0.65-2.10 mM (correspondingto 496.5-1604.2 ppm). Naringin alone at all concentrations tested didnot show detectable antimicrobial effect on bacterial species tested.Zinc Acetate alone exhibits an IC₅₀ ranging from 0.25-1.73 mM(corresponding to 45.9-317.5 ppm). The positive control Triclosan,inhibits 50% of both bacteria species in the range 0.00061-0.0076 mM(corresponding 0.18-2.2 ppm)

Further characterization was performed on planktonic oral microbes;Streptococcus sobrinus (Cariongenic—strain-SL1[CCM 6070, CNCTC 9/89](ATCC cat. #33478), Streptococcus gordonii (Arginolytic—strain V288(ATCC cat. #35105), Actinomyces viscosus) as well late colonizer (Gram−), pathogenic Aggregatibacter actinomycetemcomitans. All microbes weregrown in their respective culture media: Streptococcus sobrinus andStreptococcus gordonii in BHI broth (BD, cat #237500), Aggregatibacteractinomycetemcomitans in BHI broth supplemented with 0.5% Yeast Extract(BD, cat #210941) and Actinomyces viscosus in Trypticase Soy Broth (BD,cat #211768) supplemented with 0.5% Yeast Extract (BD, cat #210941).Bacteria were cultured overnight in 37° C., 5% CO₂ in approximately 20ml of the appropriate culture broth. The following day, the OD₆₁₀ wasdetermined and solutions were diluted to 0.2 for the assay.

Test samples (Naringin, Naringin:Zinc complex and Naringin+Zinc Acetate(combination)) were prepared as a 100 mM stock solution followed by 2fold serial dilution in 100% DMSO. Triclosan (positive control) wasprepared as a 10% stock in 100% EtOH and 3 fold serial dilutions weredone in 100% EtOH. An appropriate amount of the diluted samples weretransferred into 96 well clear plate such that the resulting finalconcentration of naringin alone, naringin:Zinc complexes, naringin+Zincacetate (combination) ranged from 2 mM to 0.0078 mM (2% DMSO) andTriclosan ranged from 0.01% to 1.7e-6% (1% EtOH), in 100 ul media perwell. For Naringin+Zinc combination, while the highest total overallconcentration was 2 mM, the exact concentration was Naringin (1.3 mM)and Zinc Acetate (0.7 mM) due to the compositions 2:1 molar ratio. ForZinc Acetate, in order to mimic the 2:1 ratio (ligand:Zinc) compositionas in the complex, the final concentration ranged from 0.7 mM to 0.0027mM in 2% DMSO.

A 100 μl aliquot of bacterial culture at OD₆₁₀ 0.2 were introduced tothe wells with the test samples. The wells were thoroughly mixed andplates incubated overnight in a 37° C. incubator with or without 5% CO₂depending upon the bacterial species. Additionally, co-cultures ofvarious bacterial species (S.s+A.a; A.a+A.v; S.s+A.v; S.g+A.v; S.g+S.$)were also performed. In such case, 50 μl of each bacterial culture wasutilized for a total volume of 100 μl per well.

After approximately 12-13 hours of treatment, the plate was read at 610nm using an Envision plate reader (PerkinElmer, Waltham, Mass.). The %inhibition relative to control was determined as follows:

${\%\mspace{14mu}{Inhibition}} = {{100\;\%} - \left( {\frac{{Treated}\mspace{14mu}{Sample}}{Control} \times 100\%} \right)}$

Naringin:Zinc complex exhibits a synergistic antimicrobial effect onboth Gram+ and Gram− planktonic oral bacterial species, cultured aloneor as co-cultures with another bacterial species. See FIGS. 11-18 andTables 6-12.

TABLE 7 Effects of treatments on the % inhibition of S. gordonii and S.sobrinus. The concentration of sample treatment chosen to mimic thestoichiometric ratio of 2:1 (Naringin to Zinc). % Inhibition Naringin(0.325 mM) + Naringin:Zinc Zinc acetate Zinc Acetate Naringin aloneComplex (0.175 mM) alone Bacterial species (0.5 mM) (0.5 mM) (0.5 mMtotal) (0.175 mM) S. gordonii  0.96 ± 2.82 66.35 ± 0.00 6.62 ± 2.1713.51 ± 10.18 S. sobrinus −2.64 ± 2.54 84.30 ± 3.67 7.33 ± 4.23 −1.05 ±3.10  S. gordonii/S. sobrinus −6.84 ± 1.19  74.8 ± 3.09 1.05 ± 6.17 1.55± 0.24

The Naringin-Zinc complex when added to single or co-cultured bacteriumresulted in a synergistic inhibition of both single species and ofco-cultures of bacteria. Naringin alone, in combination with Zincacetate or Zinc acetate alone showed little to no detectable inhibition.Additionally, the complex appeared to exhibit differential effects ondifferent species of bacteria. In this case, the complex at 0.5 mM ismore effective as an antimicrobial agent against S. sobrinus (84.30%)than S. gordonii (66.35%) and co-cultures of both resulted in 74.8%inhibition.

TABLE 8 Effects of treatments on the % inhibition of S. gordonii and S.sobrinus. The concentration of the sample treatment was chosen to mimicthe stoichiometric ratio of 2:1 (Naringin to Zinc). % InhibitionNaringin (0.325 mM) + Naringin:Zinc Zinc acetate Zinc Acetate Naringinalone Complex (0.175 mM) alone Bacterial species (0.5 mM) (0.5 mM) (0.5mM total) (0.175 mM) S. gordonii  0.96 ± 2.82 66.35 ± 0.00 6.62 ± 2.17 13.51 ± 10.18 A. viscosus −3.50 ± 4.10 34.13 ± 0.73 3.96 ± 0.88 −1.84 ±5.86 S. gordonii/A. viscosus −2.39 ± 5.68  29.9 ± 0.67 0.32 ± 2.17 −0.74± 6.34

The naringin:Zinc complex when added to single or co-cultured bacteriumresulted in a synergistic inhibition of both single species and ofco-cultures of bacteria. Naringin alone, in combination with ZincAcetate or Zinc Acetate alone showed little to no detectable inhibition.Additionally, the complex appeared to exhibit differential effects ondifferent species of bacteria. In this case, the complex at 0.5 mM ismore effective as an antimicrobial agent against S.gordonii (66.35%)than A. viscosus (34.13%) and co-cultures of both exhibited 29.9%inhibition.

TABLE 9 Effects of various treatments on the % inhibition of S. sobrinusand A. Viscosus. % Inhibition Naringin (0.325 mM) + Naringin:Zinc Zincacetate Zinc Acetate Naringin alone Complex (0.175 mM) alone Bacterialspecies (0.5 mM) (0.5 mM) (0.5 mM total) (0.175 mM) S. sobrinus −2.64 ±2.54 84.30 ± 3.67 7.33 ± 4.23 −1.05 ± 3.10 A. viscosus −3.50 ± 4.1027.07 ± 1.88 7.78 ± 1.88 −1.84 ± 5.86 S. sobrinus/A. viscosus −3.79 ±7.15  29.9 ± 0.67 0.32 ± 2.17  2.46 ± 6.58

The naringin:Zinc complex when added to single or co-cultured bacteriumresulted in a synergistic inhibition of both single species and ofco-cultures of bacteria. Naringin alone, in combination with Zincacetate or Zinc acetate alone showed little to no detectable inhibition.Additionally, the complex appeared to exhibit differential effects ondifferent species of bacteria. In this case, the complex at 0.5 mM ismore effective as an antimicrobial agent against S. sobrinus (84.30%)than A. viscosus (27.07%) while co-cultures showed 29.9% inhibition.

TABLE 10 Effects of various treatments on the % inhibition of S.actinomycetemcomitans and A. Viscosus. The concentration of the sampletreatment was chosen to mimic the stoichiometric ratio of 2:1 (Naringinto Zinc). % Inhibition Naringin (0.325 mM) + Naringin:Zinc Zinc acetateZinc Acetate Naringin alone Complex (0.175 mM) alone Bacterial species(0.5 mM) (0.5 mM) (0.5 mM total) (0.175 mM) A. actinomycetemcomitans 5.01 ± 1.69 56.47 ± 4.23 20.87 ± 0.42  29.24 ± 0.42 A. viscosus −3.50 ±4.10 27.07 ± 1.88 7.78 ± 1.88 −1.84 ± 5.86 A. actinomycetemcomitans/−0.03 ± 2.02 37.52 ± 4.03 8.05 ± 2.01  4.13 ± 6.89 A. viscosus

The naringin:Zinc complex when added to single or co-cultured bacteriumresulted in a synergistic inhibition of both single species and ofco-cultures of bacteria. Naringin alone, in combination with ZincAcetate or Zinc Acetate alone showed little to no detectable inhibition.Additionally, the complex appeared to exhibit differential effects ondifferent species of bacteria. In this case, the complex at 0.5 mM ismore effective as an antimicrobial agent against A.actinomycetemcomitans (56.47%) than A. viscosus (27.07%) whileco-cultures of both resulted in 37.52% inhibition.

TABLE 11 Effects of various treatments on the % inhibition of S.sobrinus and A. actinomycetemcomitans. The concentration of the sampletreatment was chosen to mimic the stoichiometric ratio of 2:1 (Naringinto Zinc). % Inhibition Naringin (0.325 mM) + Naringin:Zinc Zinc acetateZinc Acetate Naringin alone Complex (0.175 mM) alone Bacterial species(0.5 mM) (0.5 mM) (0.5 mM total) (0.175 mM) S. sobrinus −2.64 ± 2.54 84.30 ± 3.67 7.33 ± 4.23 −1.05 ± 3.10 A. actinomycetemcomitans 5.01 ±1.69 56.47 ± 4.23 20.87 ± 0.42  29.24 ± 0.42 A. actinomycetemcomitans/0.33 ± 3.48  79.1 ± 0.54 9.42 ± 2.95  6.58 ± 5.36 S. sobrinus

The naringin:Zinc complex when added to single or co-cultured bacteriumresulted in a synergistic inhibition of both single species and ofco-cultures of bacteria. Naringin alone or in combination with ZincAcetate or Zinc Acetate alone showed little to no detectable inhibition.Additionally, the complex appeared to exhibit differential effects ondifferent species of bacteria. In this case, the complex at 0.5 mM wasmore effective as an antimicrobial agent against S. sobrinus (84.30%)than A. actinomycetemcomitans (56.47%) while co-cultures exhibited 79.1%inhibition.

As a positive control for the antimicrobial assay, the same species ofbacteria cultures were treated with Triclosan in the range of0.01%-1.7e-6% in 1% EtOH final concentration. The IC₅₀ was determinedusing GraphPad Prism version 6 software to be in the range of0.00010-0.00027% (1-2.7 ppm).

The findings here show that the antimicrobial efficacy of naringin canbe enhanced with the presence of metal zinc within as a naringin:Zinccomplex or as a combination of Zinc and naringin. Surprisingly, theanti-microbial efficacy is enhanced with the naringin:Zinc complexes ofthe present invention, which are, at least partially, characterized byhaving 2:1 stoichiometric ratio of naringin to Zinc.

Antimicrobial characterization was also performed on mixed culturesolutions having at least five microbial species present. A mixedculture containing Actinomyces viscosus (A. viscosus, A.v) (ATCC,#43146), Lactobacillus casei (L. casei, L.c) (ATCC #334), Streptococcusoralis (S. oralis, S.o) (ATCC #35037), Fusobacterium nucleatum (F.nucleatum, F.n) (ATCC #10953) and Veilonella parvula (V. parvula, V.p)(ATCC #17745) was maintained in a specialized complex medium (ModifiedBHI II Medium) in a continuous culture chemostat at 37° C. Approximately10 ml of the culture was removed and the OD₆₁₀ was determined. The assaywas performed using cultures at two concentrations; 0.03 and 0.1 atOD₆₁₀. Test samples (Naringin, Naringin:Zinc complex and Naringin+ZincAcetate (combination)) were prepared as a 100 mM stock solution followedby 2 fold serial dilution in 100% DMSO. Triclosan (positive control) wasprepared as a 10% stock in 100% EtOH and 3 fold serial dilutions wereperformed in 100% EtOH. Appropriate amount of the diluted samples weretransferred into 96 well clear plate such that the resulting finalconcentration of Naringin alone, Naringin:Zinc complexes, Naringin+ZincAcetate (combination) ranged from 2 mM to 0.0078 mM (2% DMSO) andTriclosan ranged from 0.01% to 1.7e-6% (1% EtOH), in 100 ul BH II media(Alvarez G. et al.; AMB Express; 2013, 3(1), doi: 10.1186/2191-0855-3-1)per well. For Naringin+Zinc acetate combination, while the highest totaloverall concentration was 2 mM, the exact concentration was Naringin(1.3 mM) and Zinc Acetate (0.7 mM) due to the compositions 2:1 molarratio. For Zinc Acetate, in order to mimic the 2:1 ratio (ligand:Zinc)composition as in the complex, the final concentration range from 0.7 mMto 0.0027 mM in 2% DMSO was used instead.

A 100 μl of bacterial culture at chosen OD₆₁₀ (0.03 or 0.1) wasintroduced into the wells with the test samples. The wells werethoroughly mixed and plates incubated overnight in a 37° C. incubatorwith or without 5% CO₂. After approximately 12-13 hours of treatment,the plate was read using Envision plate reader (Serial #1040984),absorbance at 610 nm. The % inhibition relative to control wasdetermined as described for previously described antimicrobialprotocols, supra.

Using mixed species cultures, we observed that Naringin:Zinc complexresulted in a synergistic inhibition in comparison to naringin+Zinccombination and Zinc acetate alone regardless of whether the cultureswere incubated with or without 5% CO₂ (see FIG. 17, FIG. 18, and Table12). The antimicrobial efficacy of the complex was more potent againstcultures incubated in the absence of 5% CO₂ (see Table 12).

TABLE 12 Summary of the effects of various treatments on mixed culturesolutions. % Inhibition Naringin (0.1625 mM) + Naringin:Zinc Zincacetate Zinc Acetate Bacterial species and Naringin alone Complex(0.0875 mM) alone culturing conditions (0.25 mM) (0.25 mM) (0.25 mMtotal) (0.0875 mM) Mixed species incubated 3.512 ± 1.53 48.244 ± 0.31−1.67 ± 5.20 −1.46 ± 0.0 in 37° C., 5% CO2 Mixed species incubated 3.099± 3.31  81.70 ± 4.63  3.10 ± 8.60  18.07 ± 2.65 in 37° C.; Air

We observed that in the mixed species cultures irrespective of whetherthe cultures were incubated aerobically or anaerobically, the overalloutcome is a synergistic antimicrobial effect in the presence of thecomplex, not their individual parts alone or in combination. However, inthe aerobic condition, the antimicrobial efficacy of the complex isalmost doubled that of the cultures in the anaerobic condition (81.7% vs48.2%) for the same concentration tested.

For a positive control, the mixed species cultures were treated withTriclosan in the range of 0.01%-1.7e-6% in 1% EtOH. The IC₅₀ wasdetermined, using GraphPad Prism version 6 software, to be in the rangeof 0.00014-0.00022% (1.4-2.2 ppm) (data not shown).

Example 14—Tissue Zinc Uptake Analysis

EpiGingival™ tissue models used for the zinc uptake study were purchasedfrom MatTek Corporation (Ashland, Mass., cat # Gin-606). All sampleswere prepared in a final concentration of 1% DMSO in 7% of PropyleneGlycol (PG). For example, 1 mM of naringin:Zinc complex was prepared ina final concentration of 1% DMSO and 7% PG; naringin and Zinc acetatecombination was prepared such that the total concentration was 1 mM butconsisted of 0.67 mM of naringin and 0.33 mM Zinc acetate in 1% DMSO and7% PG. Zinc acetate was prepared at 0.33 mM to mimic the amount of zincthat is present in 1 mM of naringin:Zinc complex. For the vehicle onlycontrol, 1% DMSO in 7% PG was used. All samples were equilibrated to 37°C. prior to testing.

Zinc uptake studies were performed using EpiGingival™ tissue models fromMatTek Corporation. Briefly, EpiGingival™ tissue inserts wereequilibrated overnight in 1 ml of the EpiGingival™ assay media (suppliedby MatTek Corporation). For testing, working with one insert at a time,the insert was removed and placed inside a clean 6 well plate. Then, 1ml of the test sample was carefully removed and introduced to the insideof the insert and allowed to incubate for 2 minutes. Next, the sampleswere removed using an aspirator and the tissue rinsed with 1 ml of warmwater (37° C.). This was repeated for 2 more times for a total of 3washes. At the end of the last wash making sure to remove as much of thewater as possible, the tissue was disengaged from the insert with thetip of a sterile forceps, and transferred into a clean 15 ml Falcontube. The process was repeated until all the tissues were treated.Experiments were performed three times with each test sample assayed induplicate.

Digestion of Tissues: Briefly, 0.5 ml of a mixture of 7:3 HCl/HNO₃ wasintroduced into a 15 ml Falcon tube containing one of the treated tissuesamples and allowed to digest overnight. The following day, 4.5 ml ofwater was added to each of the digested tissue samples, thoroughly mixedand centrifuged at 4,000 RPM for 10 minutes at room temperature. Theclear supernatant (4.5 ml) was transferred into a clean, labeled Falcontube and submitted for analytical analysis of Zinc.

Average zinc uptake (ppm) and the standard deviation of samples werecalculated using Microsoft Excel 2010. A one way ANOVA was done todetermine the p value. A plot of the average zinc (ppm) against thevarious treatments was generated (see FIG. 19).

Naringin:Zinc complex (Product 5) exhibited a significant zinc uptake incomparison to the naringin+Zinc acetate combination at the same molarconcentration. By contrast, zinc uptake by Product 5 was determined tobe not significantly different from that by Zinc Acetate alone at thesame molar concentration. The zinc uptake and the antimicrobialcharacteristics taken together, without being bound to theory, suggestthe observed synergistic antimicrobial effect may be a reflection of thecomplex as a whole rather than zinc acetate alone.

TABLE 13 Summary of Zinc uptake from EpiGingival ™ tissue models after 2minute treatment with Naringin:Zinc complex (Product 5), combination ofnaringin and Zinc acetate and Zinc acetate alone. Preparation 1, 2 and 3refers to three different synthesized batches of Product 5. Eachexperiment was performed in duplicate (N1 and N2); Zn refers to Zinc inparts per million (ppm). Zn (ppm) Preparation Preparation Preparation 12 3 Ave Samples N1 N2 N1 N2 N1 N2 (ppm) STDEV Product 5 0.94 0.82 1.221.31 1.07 1.2 1.09 0.19 Naringin + 0.93 0.86 0.91 0.81 0.75 0.76 0.840.08 Zinc acetate Zinc 0.96 0.92 0.82 0.81 0.84 0.85 0.87 0.06 acetateonly Vehicle 0.2 0.22 0.3 0.24 0.23 0.2 0.23 0.04 only

Example 15—Synthesis Method 6

Two moles Naringin (1.21 g.) and one mole ZnO (0.081 g.) was mixed in100 ml H₂O. The solution was heated to 80° C. for 4 hours with vigorousstirring. A yellowish powder was formed (1.102 g., 90% yield). Thepowder was washed with hot water (3×100 ml). Mixing 21.4 mg of thepowder in 10% HNO₃ produced a clear solution. The product, labeled asProduct 7, was characterized using FTIR, ¹H-NMR and Diffusion OrderedSpectroscopy.

Example 16—Wound Healing

Cell Culture: Spontaneously immortalized human keratinocyte line, HaCaTcells (AddexBio, San Diego, Calif., cat # T0020001) were cultured in 25cm² vented, Falcon canted tissue culture flasks (VWR, Radnor, Pa., cat#353108) using complete DMEM media (Life Technologies, Grand Island,N.Y., cat #11965-092) supplemented with 10% characterized Hyclone FBS(VWR, Radnor, Pa., cat #1677-014) and 1× Penicillin—Streptomycin (LifeTechnologies, Grand Island, N.Y., cat #15140122). The cultures wereincubated at 37° C. and 5% CO₂ until the cells reached around 70-80%confluence. Cells were then detached with Trypsin—EDTA solution (Sigma,St. Louis, Mo., cat # T3924) and used either for propagation or inexperimentation.

Assay sample preparation: Samples were prepared in a final concentrationof 1% DMSO in complete media. The following were prepared—naringin at0.1 mM, naringin:zinc complex at 0.15 mM, Zinc acetate at 0.05 mM andnaringin+Zinc acetate combination at a final concentration of 0.15 mM.1% DMSO in complete media was used as a vehicle control and media aloneas the positive control. Each treatment was done in quadruplets and theaverage was taken. Two different batches of product 5 were tested at0.15 mM, the average % Growth Relative to day 0 (d0) was determined andpresented in FIG. 20.

Preparation of wound: HaCaT cells at around 70-80% confluence weretreated with 1 ml of Trypsin-EDTA solution and incubated at 37° C., 5%CO₂ until the cells detached from the flask. 5 ml of the complete mediawere added to stop further trypsinization of the cells. The cellsuspensions were transferred into a clean 50 ml Falcon centrifuge tubeand spun down at 1500 RPM for 5 minutes to pellet the cells. Pelletedcells were re-suspended in 50 ml of fresh complete media and 1 ml of thecell suspensions was transferred into each well of a Falcon 35304724-well plate (VWR, Radnor, Pa.). The plates were incubated at 37° C.,5% CO2 until cells reached around 90% confluence.

On the day of the assay, a “crossed-shaped image” was asepticallycreated onto the bottom of each of 24 well plate cell monolayers using asterile p1000 pipette tip to simulate a “wound” (see FIG. 21). The cellswere washed twice with 1 ml of plain media with gentle shaking for aminute and then refreshed with 1 ml of appropriate test media.

Quantifying cell migration/wound healing: To explore the effects ofvarious actives on cell migration/wound healing, the size of the freshlygenerated wound (the gap size) was determined following treatment usingan inverted microscope (Olympus 1X71, Olympus Scientific SolutionsAmericas, Waltham, Mass.) and Cellsens Dimension 1.11 (OlympusCorporation, Waltham, Mass.) software and monitored until the gap closedcompletely. Three different measurements were taken for each arm of the“crossed” wound image. A total of 12 measurements were taken for each ofthe 24 well (3 readings from the top, 3 from the bottom, 3 from theright and remaining 3 from the left) (see FIG. 21). The mean of each armwas determined for each day of the study until the wound heal and no gapexisted. The % of cell growth relative to d0 (day 0) for each treatmentwas calculated as follows:

${\%\mspace{14mu}{Growth}\mspace{14mu}{relative}\mspace{14mu}{to}\mspace{14mu} d\; 0} = {\frac{\begin{matrix}{{{measurement}\mspace{14mu}{of}\mspace{14mu}{gap}\mspace{14mu}{at}\mspace{14mu} d\; 0} -} \\{{measurement}\mspace{14mu}{of}\mspace{14mu}{gap}\mspace{14mu}{post}\mspace{14mu}{treatment}}\end{matrix}}{{measurement}\mspace{14mu}{of}\mspace{14mu}{gap}\mspace{14mu}{at}\mspace{14mu} d\; 0} \times 100\%}$

Results: As shown in FIG. 20, treatment with Product 5 resulted in anenhanced “wound healing” when compared to naringin treatment alone, Zincacetate alone as well as the combination of naringin and Zinc acetate.Naringin treatment alone was not significantly different from that ofZinc acetate, the combination of both naringin and Zinc acetate, vehiclealone as well as the media treatment. Comparing media to vehicle aloneshowed inhibition of growth in vehicle treated wells (possibly due tothe presence of DMSO). Surprisingly, cells treated with Product 5 wereable to recover much better than individual components alone or incombination. Similar findings were observed with 0.20 mM of Product 5(data not shown).

While the present invention has been described with reference toembodiments, it will be understood by those skilled in the art thatvarious modifications and variations may be made therein withoutdeparting from the scope of the present invention.

What is claimed is:
 1. A method of preparing an oral care compositioncomprising a naringin:Zn complex, wherein said naringin:Zn complex has a2:1 naringin to zinc molar ratio; wherein the oral care composition maybe any of the following selected from the group consisting of: atoothpaste or a dentifrice, a mouthwash or a mouthrinse, a topical oralgel and a denture cleanser.
 2. The method of claim 1, wherein thenaringin:Zn complex has a melting point above 230° C.
 3. The methodcomplex of claim 1, wherein the naringin:Zn complex has a diffusioncoefficient between 2.8e-11 to 3.2e-11 m²/s in DMSO solution at 25° C.4. The method of claim 1, wherein the oral care composition furthercomprises one or more agents selected from diluents, bicarbonate salts,pH modifying agents, surfactants, foam modulators, additional thickeningagents, humectants, sweeteners, flavorants, pigments, antibacterialagents, anticaries agents, fluoride ion sources, anticalculus or tartarcontrol agents, and mixtures thereof.
 5. The method of claim 1, whereinthe composition is prepared using a pH between 7-10.
 6. The method ofclaim 1, wherein mixing of naringin and zinc is performed at atemperature between 65-85° C.
 7. The method of claim 1, whereinpreparation of said complex comprises the steps of: a. mixing naringinin methanol; b. adding a source of zinc; c. adjusting the pH of thesolution to 10.0; d. incubating the reaction; and e. optionallyisolating said complex.
 8. The method of claim 1, wherein preparation ofsaid complex comprises the steps of: a. mixing naringin in water; b.heating the mixture to 70° C.; c. adding a source of zinc; d. adjustingthe pH of the solution to 10.0; e. incubating the reaction; and f.optionally isolating said complex.
 9. The method of claim 1, whereinpreparation of said complex comprises the steps of: a. mixing naringinin water; b. heating the mixture to 70° C.; c. adjusting the pH of thesolution to 10.0; d. adding a source of zinc; and e. optionallyisolating said complex.
 10. The method of claim 1, wherein preparationof said complex comprises the steps of: a. mixing naringin in water; b.heating the mixture to 70° C.; c. adding a source of zinc; d. adjustingthe pH of the solution to 7.0; and e. optionally isolating said complex.11. The method of claim 1, wherein preparation of said complex comprisesthe steps of: a. mixing naringin in propylene glycol at 70° C.; b.adjusting the pH of the solution to be between 9.0-10.0; c. adding asource of zinc in propylene glycol at 70° C.; and d. optionallyisolating said complex.
 12. The method of claim 7, wherein said Znsource is selected from zinc acetate, zinc oxide, zinc chloride, zinclactate, zinc citrate, or zinc nitrate.
 13. The method of claim 7,wherein said Zn source is zinc acetate.
 14. The method of claim 1wherein ZnO is used as a source of Zn for the reaction.
 15. The methodof claim 14, said method comprises the steps of: a. mixing naringin andZnO in water; b. heating the mixture; c. incubating the mixture; and d.optionally isolating said complex.